Research article Received: 22 December 2014,
Revised: 9 February 2015,
Accepted: 19 February 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3462
UHPLC-MS method for determination of gambogic acid and application to bioavailability, pharmacokinetics, excretion and tissue distribution in rats Zhifen Zhenga, Wanglu Oua, Xinshi Zhangb, Yongzhi Lic and Yujuan Lia* ABSTRACT: A sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was developed for determination of gambogic acid (GA) in rat plasma, urine, bile and main tissues. GA was separated on an Agilent Zorbax XDB–C18 column (50 × 2.1 mm, 1.8 μm) with gradient mobile phase at the flow rate of 0.2 mL/min. The detection was performed by negative electrospray ionization with multiple reaction monitoring mode. The calibration curves of GA were linear between 1.0 and 1000 ng/mL in rat plasma and bile and between 1.0 and 500 ng/mL in urine and tissues. The lowest limit of quantification for all matrices was 1.0 ng/mL. Both accuracy and precision of the assay were satisfactory. This validated method was firstly applied to bioavailability (BA), pharmacokinetics, excretion and tissue distribution in rats. The BAs of GA (40 and 80 mg/kg) in rats were 0.25 and 0.32%, respectively. GA was distributed extensively in rats after oral administration and exhibited the highest level in liver. GA reached the cumulative excretion amount of 25.3 ± 1.7 μg in bile and 0.275 ± 0.08 μg in urine after i.g. 80 mg/kg to rats at 24 h. The present results would be helpful for further clinical use of GA as a potential anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: gambogic acid; UHPLC-MS; bioavailability; excretion; tissue distribution
Introduction Gambogic acid (GA, Fig. 1) is a natural caged polyprenylated xanthone (Hou and Xiao, 2011; Song et al., 2007; Yang et al., 2013; Li et al., 2008). It was isolated from Gamboge (the resin of Garcinia hanburyi Hook. F., a traditional Chinese medicine). Gamboge possesses anti-inflammatory, anti-viral and anti-cancer effects (Xu and Yang, 2013; Yen et al., 2012; Wen et al., 2014; Kong et al., 1996). As a promising natural anti-cancer candidate, GA’s activity in numerous types of cancer cells has been well demonstrated in vivo and in vitro, including leukemia, lung, liver, SH-SY5Y neuroblastoma, breast, colorectal, U87 glioma, ovarian and gastrointestinal cancer cells (Chen et al., 2014; Zhang et al., 2014; Qi et al., 2014; Duan et al., 2014; He et al., 2013; Wang and Yuan, 2013; Rahman et al., 2013; Li et al., 2012; Zou et al., 2012). In addition to significant anti-tumor effects, GA also exhibits anti-inflammatory, detoxification and parasiticide effects (Yen et al., 2012; Wen et al., 2014; Rahman et al., 2013). However, although GA shows various pharmacological activities, information regarding the in vivo fate of GA is still limited. There has been no report for GA bioavailability, excretion or tissue distribution with a sensitive method either in humans or in animals. To date, several techniques have been employed for analysis of GA in Gamboge (only in the crude herb, not in biomatrices) for quality assessment, such as high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection (Song et al., 2007; Li et al., 2008), capillary electrophoresis with UV detection (Lopez-Montes et al., 2007) and HPLC with evaporative light-scattering detection (Yang et al., 1999). HPLC tandem electrospray ionization mass spectrometry (HPLC-ESI/MS) has
Biomed. Chromatogr. 2015
been utilized for the identification of xanthones in Gamboge herb (Zhou et al., 2008), and for structural identification for GA metabolites in rats or human (Feng et al., 2007; Yang et al., 2010). A HPLC-UV method has been reported to study pharmacokinetics in dog plasma with a lowest limit of quantification (LLOQ) of 156 ng/mL (Hao et al., 2007a, 2007b). Ding et al. (2007) developed a liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry with single ion monitoring method for the determination of GA in human plasma with LLOQ of 31 ng/mL. In above three reports the administration route was via intravenous injection. These methods might be sensitive enough for the quantification of GA in biosamples after intravenous injection. Recently, Hua et al. (2014) quantified GA and gambogenic acid in rat plasma after oral administration of Garcinia hanburyi extracts by LC-MS. Bioavailability (BA), excretion and tissue distribution for GA were not reported in the above paper.
* Correspondence to: Y. Li, School of Life Science, Beijing Institute of Technology, Beijing 100081, China. Email: [email protected] a
School of Life Science, Beijing Institute of Technology, Beijing 100081, China
b
Hebei North University, Zhangjiakou 075000, China
c
China Astronaut Research and Training Centre, Beijing 100094, China Abbreviations used: BA, bioavailability; GA, gambogic acid; MRM, multiple reaction monitoring.
Copyright © 2015 John Wiley & Sons, Ltd.
Z. Zheng et al. generation of precursor and fragment ions. Optimum parameters were as follows: nebulizer (GS1), heater (GS2) and curtain gas flow rates, 40, 50 and 10 arbitrary units, respectively; heater gas temperature, 500 °C; ion spray needle voltage, 4500 V; declustering potential, 60 and 50 V for GA and IS, respectively; collision energies, 23 and 28 V for GA and IS, respectively; collision potential, 11 and 8 V for GA and IS, respectively; cell exit potential, 18 and 10 V for GA and IS, respectively.
Figure 1. Chemical structures of gambogic acid (GA) (A) and IS (IS).
It is known that different bio-matrices need different fully validated methods. To our knowledge, HPLC-MS methods are not available for the determination of GA in urine, bile or tissues of experimental animals or human. Few studies have been carried out for evaluating urinary and biliary excretion or tissue distribution of GA in rats with HPLC-MS. In addition, reports on the dosedependent pharmacokinetics of single GA in rats after oral administration have been not proposed elsewhere. Further, the oral bioavailability of GA was not previously available. Therefore, the present study aimed to develop a reliable and sensitive ultra-HPLC-MS method with an LLOQ of 1.0 ng/mL for quantification of GA in rat plasma, urine, bile and main tissues. It has been applied to the investigation of BA, pharmacokinetics, excretion and tissue distribution in rats for the first time. The expected results might supply more in vivo information for further clinical application of GA.
Calibration standards and quality control sample preparation A 1.0 mg aliquot of GA was dissolved in 1.0 mL of methanol to give the concentration of 1.0 mg/mL as the stock solution. Working solutions were prepared by dilution of the stock solution with methanol–water (9:1, v/v, 90% methanol) to obtain series of working concentrations. All of the solutions were stored at 4 °C before use. A 1.0 mg aliquot of phenytoin sodium (IS) was dissolved in 1.0 mL of methanol to give the concentration of 1.0 mg/ mL as the IS stock solution. IS solution was finally diluted to 20 ng/mL with acetonitrile and kept at 4 °C until use. The calibration standards (1, 2, 5, 10, 50, 100, 500 and 1000 ng/mL) were prepared by spiking blank plasma or bile with appropriate amounts of working solutions to yield a final concentration range of 1.00–1000 ng/ mL. For urine and tissue calibration curves, calibration standards (1, 2, 5, 10, 50, 100 and 500 ng/mL) were prepared by spiking blank urine or tissues with appropriate amounts of working solutions to yield a final concentration range of 1.00–500 ng/mL. Quality control (QC) samples were prepared with at three concentration levels of 1, 80 and 800 ng/mL for rat plasma and bile, and 1, 80 and 400 ng/mL for rat urine and tissues.
Sample preparation
Experimental Chemicals and reagents GA (99.1%, purity) was supplied by Shanghai Yuanye Biotech Company (Shanghai, China). Phenytoin sodium (98.9%, purity, used as internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The chemical structures of GA and IS are shown in Fig. 1. Methanol and acetonitrile were of chromatographic grade and supplied by Thermo Fisher Scientific (California, USA). Ammonium acetate was of analytical grade from Yili Company (Beijing, China). Distilled water, prepared using a Milli-Q water Millipore purification system (Molsheim, France), was used throughout the study.
A 50 μL aliquot of plasma samples (or blank plasma) was mixed with 5 μL of 90% methanol (or working solutions) and 150 μL of IS solution in vials. The mixture was vortexed for 2 min. The samples were centrifuged at 10,000 g for 10 min. A 10 μL aliquot of supernatant was injected onto the chromatographic column for analysis. For urine, bile and tissue homogenates samples, 10 μL of samples (or blank urine, bile, tissue homogenate) was transferred to vials and mixed with 5 μL of 90% methanol (or working solutions). After 150 μL of IS solution was added, the mixture was thoroughly vortexed for 2 min. The samples were centrifuged at 10,000 g for 10 min. A 10 μL aliquot of supernatant was injected onto the chromatographic column for analysis.
Method validation LC-MS/MS conditions The instrument used in the present experiment mainly contained an Agilent 1290 UHPLC system (Agilent Technologies, Mountain View, California, USA), TM an Applied Biosystems Sciex 4000 Q-trap mass spectrometer via a Turbo Ion Spray ionization interface (Concord, Ontario, Canada) and an Infinity sampler G4226A (Agilent, California, USA). Applied Biosystems Analyst software version 1.5.1 package (Applied Biosystems, California, USA) was used to control the UHPLC-MS system and for data acquisition and processing. An Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 μm, Agilent Technologies, California, USA) was used for chromatographic separation. The mobile phase consisted of a mixture of 0.06% ammonium acetate in water (A) and 0.06% ammonium acetate in acetonitrile–methanol (1:1, v/v) (B). The gradient elution was programmed as follows: 0.0–0.4 min 80% A; 0.4–1.0 min 20% A; 1–4.0 min, 5% A; 4.0–5.0 min, 80% A. The flow rate was 0.2 mL/min and the column temperature was set at 25 °C. The mass spectrometer was operated in the negative electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) at unit resolution. Nitrogen was used as the nebulizer, heater and curtain gas as well as the collision-activated dissociation gas. The precursor-to-product ion transitions were monitored at m/z 627.3 → 583.2 for GA and m/z 251.2 → 101.9 for IS. Mass spectrometer parameters were tuned to maximize the
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The method was validated according to the Food and Drug Administration guidance for bioanalytical method validation, including linearity, selectivity, precision and accuracy, recovery and stability (US Food and Drug Administration, 2001). The matrix effect was also evaluated following the published method (Matuszewski et al., 2005). 2 Linearity was assessed by weighted linear regression (1/x , where x is the concentration of GA in rat plasma, bile, urine or tissues) of analyte–internal standard peak area ratios (y) based on three independent calibration curves prepared on each of three separate days. Selectivity was tested by comparison of blank matrices from six individual rats with corresponding spiked matrices. At the same time, spiked solutions with calibration concentration levels were used to determine calibration curve. QC samples at three concentrations in six replicates were analyzed during the same day using the same calibration curve to determine the intra-day precision. All of the samples mentioned above were analyzed for three continuous days to define inter-day precision. Precision was expressed as the relative standard deviation (RSD). Accuracy was calculated as the relative error (RE). The lowest limit of quantitation (LLOQ) is the lowest concentration of analyte on the calibration curve, which should be quantified reliably with an acceptable accuracy and precision (≤15 %; US Food and Drug Administration, 2001).
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015
Bioavailability, excretion, distribution of gambogic acid
Figure 2. Typical multiple reaction monitoring (MRM) chromatograms of (A) blank plasma; (B) blank plasma spiked with GA (10 ng/mL) and IS (20 ng/mL); (C) a real rat plasma sample at 4 h post-dosing; (D) a real rat bile sample within 0–4 h post-dosing; (E) a real rat urine sample within 12–24 h post-dosing; (F) a real rat liver sample at 1 h post-dosing. Peak I, GA; peak II, IS.
Table 1. Calibration range, equation, correlation coefficient and lowest limit of quantification (LLOQ) of gambogic acid (GA) in all biological matrices Matrix
Calibration range (ng/mL)
Calibration equation
Correlation coefficient (r)
LLOQ (ng/mL)
Plasma Bile Urine Heart Liver Spleen Lung Kidney Brain
1.00–1000 1.00–1000 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500
y = 0.429x + 0.032 y = 0.043x + 0.046 y = 0.030x + 0.027 y = 0.031x + 0.029 y = 0.024x + 0.018 y = 0.018x + 0.062 y = 0.048x + 0.026 y = 0.023x + 0.031 y = 0.003x + 0.022
0.9959 0.9964 0.9961 0.9952 0.9949 0.9961 0.9980 0.9979 0.9980
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Table 2. Precision and accuracy for determination of GA in rat plasma, urine, bile and liver by UHPLC-MS Method (n = 6) Matrix
Concentration ng/mL
Plasma
Bile
Urine
liver
1.00 80.0 800.0 1.00 80.0 800.0 1.00 80.0 400.0 1.00 80.0 400.0
Precision (RSD, %)
Accuracy (RE, %)
Intra-day
Inter-day
Intra-day
Inter-day
3.4 2.8 7.1 2.7 9.2 2.6 4.6 7.4 1.4 6.6 4.8 3.6
9.0 1.7 1.8 2.8 3.4 8.7 9.1 2.8 3.3 4.9 1.6 2.7
6.8 –2.1 3.2 2.4 8.6 –1.9 –4.7 8.8 –5.6 4.7 –2.1 –0.9
–3.9 –3.4 6.8 7.7 –5.6 8.9 3.2 4.6 –6.7 2.1 –3.4 7/9
Note, accuracy and precision for other tissues are not listed here.
Biomed. Chromatogr. 2015
Copyright © 2015 John Wiley & Sons, Ltd.
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2.2
–2.9 3.4
–8.1 –1.7
–0.8
6.2
2.9
5.1
–0.9 2.1 3.2
1.8 4.5
2.6 –7.7
Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed urine samples at 25 °C (RE %) 8h Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed liver samples at 25°C (RE %) 8h 2.6 –3.1 –5.8
–4.9 2.6 3.4
2.1 –2.6
2.6 –6.7
3.4
2.9 –3.1
1.9
Liver
Urine 2.8 –6.4 1.6
800 80.0 1.00
–8.1
Conditions Matrix Spiked concentration (ng/mL)
Copyright © 2015 John Wiley & Sons, Ltd.
Note, stabilities for other tissues are not listed here.
The rats were put into metabolism units to collect urine samples at 4, 8, 12, 24, 36, 48 and 60 h after dosing. Rats for biliary excretion experiment were first anesthetized with 3 g/kg of chloral hydrate and were surgically implanted with a cannula in the bile duct. Bile samples were collected at 4, 8, 12 and 24 h post-ingestion. Blank urine and bile was collected before dosing. The volumes of urine and bile samples were recorded. The urine and bile samples were centrifuged at 10,000 g for 10 min and then all supernatant was kept at 20 °C until use.
Bile
Excretion after oral administration
Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed plasma samples at 25 °C (RE %) 8h Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed bile samples at 25°C (RE %) 8h
In tissue distribution experiment, rats (n = 6 for each time point) were fasted for 12 h with free access to water. After rats were orally administered 80 mg/kg GA, main tissues (heart, liver, spleen, lung, kidney and brain) were collected at 0.5, 1 and 4 h post-dosing and then weighed. Blank tissues samples were collected separately before ingestion. Tissues were homogenized with saline (2-fold the tissue weight) and then centrifuged at 10,000 g for 10 min to get the homogenates. The homogenate samples were stored at –20 °C until use.
Plasma
Tissue distribution after oral administration
Conditions
For two oral administration (p.o.) groups (n = 6 for each group), all animals were fasted for 12 h prior to administration with free access to water. GA was suspended in 5% (w/v) carboxy-mehtylcellulose sodium (CMC-Na) solution, and then was administrated by gastric gavage (40 and 80 mg/kg to rats, respectively). In intravenous administration (i.v.) groups (n = 6 for each group), GA dissolved in saline (containing 0.4% Tween 80 and 6% PEG300) was given via the caudal vein (4 mg/kg to rats). A blood sample (0.2 mL) was collected from orbital vein into heparinized tubes at appropriate intervals (10, 20, 30, 45 min, 1, 2, 4, 6, 9 and 12 h for p.o. group; 2, 5, 10, 30 min, 1, 2, 3, 4, 6 and 9 h for i.v. group) after dosing. A blank plasma sample was collected before dosing. All the blood samples were centrifuged at 10,000 g for 10 min and then plasma samples were stored at –20 °C until analysis.
Matrix
Oral bioavailability and pharmacokinetics
Table 3. Stability of GA in rat plasma and urine under different storage conditions determined by UHPLC-MS method (n = 6)
Animal treatment and sample collection Sprague–Dawley rats (male, 200–220 g body weight) were purchased from Beijing Military Medical Sciences Experimental Animal Co. Ltd (Beijing, China). All the rats were housed in an air-conditioned room with the temperature of 24 ± 2 °C, relative humidity of 55 ± 10% and alternating 12 h light/dark cycle. The study complied with guidelines for the Care and Use of Laboratory Animals (published by the National Institutes of Health, NIH publication no. 85-23). The experimental procedures were approved by Beijing Institute of Technology Committee on Animal Care and Use [Beijing, China. Permission Number: SYXK ( Jing) 2012-0035].
2.6
400 80.0 1.00
Spiked concentration (ng/mL)
The recovery of analytes and IS was determined by comparing the peak areas of the analytes and IS in deproteinized QC samples with those in post-deproteinization blank samples spiked at the corresponding concentrations. Freeze–thaw stability of GA in rat plasma, urine, bile and tissues was assessed using four concentration of QC samples after three freeze (–20 °C) to thaw (25 °C) cycles. Long-term stability of QC samples was evaluated after storage at –20 °C for 30 days. The stability of the analyte in the all matrices was also investigated at 25 °C for 8 h. The sample was considered as stable if the relative error was 100% mean ionization enhancement, whereas ME values < 100% indicate ionization suppression.
3.4
Z. Zheng et al.
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Bioavailability, excretion, distribution of gambogic acid 150
Methanol was first considered as elution solvent. However methanol showed poor peak shape for GA. The mixture of methanol and acetonitrile was proved to achieve proper running time and to be more economical than acetonitrile itself. After thorough optimization, the gradient elution procedure was finally chosen because it led to better peak symmetry and shorter retention times for GA and IS than isocratic elution. The retention times of GA and IS under the chromatographic conditions used were 3.58 and 2.13 min, respectively. IS was indispensable for the bio-analysis. Several internal standards, such as loureirin B, spinosin and phenytoin sodium, were tested in the present study. Spinosin showed stronger ion responses in positive than negative ion mode. Loureirin B could give a good response in both modes, while it did not give a satisfactory retention time (0.9949. The calibration equation, calibration range, LLOQ and correlation coefficient (r) in all matrices of GA are listed in Table 1. Intra-day and inter-day precisions (RSD) and accuracy (RE) for quantification of GA in rat plasma, urine, bile and liver are summarized in Table 2. Accuracy for plasma samples ranged from –6.7 to 8.8 % RE. For bile samples, accuracy was between –1.9 and 8.6 %
A
40 mg/kg,po 80 mg/kg,po
120 90 60 30 0
2
0
4
6
8
10
12
Time (h) 1000
Concentration (ng/mL)
Method development
Concentration (ng/mL)
Results and discussion
B
4 mg/kg,iv
800 600 400 200 0 0
2
4
6
8
10
Time (h) Figure 3. Mean plasma concentration–time curve of GA after oral administration to rats. (A) 40 and 80 mg/kg p.o.; (B) 4 mg/kg i.v. Data is expressed as mean ± SD (n = 6 for each time point).
RE. Accuracy for urine and liver was in the range of –5.6–8.8% RE. The intra- and inter-day precisions for plasma, urine, and bile and liver were
Revised: 9 February 2015,
Accepted: 19 February 2015
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3462
UHPLC-MS method for determination of gambogic acid and application to bioavailability, pharmacokinetics, excretion and tissue distribution in rats Zhifen Zhenga, Wanglu Oua, Xinshi Zhangb, Yongzhi Lic and Yujuan Lia* ABSTRACT: A sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS) method was developed for determination of gambogic acid (GA) in rat plasma, urine, bile and main tissues. GA was separated on an Agilent Zorbax XDB–C18 column (50 × 2.1 mm, 1.8 μm) with gradient mobile phase at the flow rate of 0.2 mL/min. The detection was performed by negative electrospray ionization with multiple reaction monitoring mode. The calibration curves of GA were linear between 1.0 and 1000 ng/mL in rat plasma and bile and between 1.0 and 500 ng/mL in urine and tissues. The lowest limit of quantification for all matrices was 1.0 ng/mL. Both accuracy and precision of the assay were satisfactory. This validated method was firstly applied to bioavailability (BA), pharmacokinetics, excretion and tissue distribution in rats. The BAs of GA (40 and 80 mg/kg) in rats were 0.25 and 0.32%, respectively. GA was distributed extensively in rats after oral administration and exhibited the highest level in liver. GA reached the cumulative excretion amount of 25.3 ± 1.7 μg in bile and 0.275 ± 0.08 μg in urine after i.g. 80 mg/kg to rats at 24 h. The present results would be helpful for further clinical use of GA as a potential anticancer drug. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: gambogic acid; UHPLC-MS; bioavailability; excretion; tissue distribution
Introduction Gambogic acid (GA, Fig. 1) is a natural caged polyprenylated xanthone (Hou and Xiao, 2011; Song et al., 2007; Yang et al., 2013; Li et al., 2008). It was isolated from Gamboge (the resin of Garcinia hanburyi Hook. F., a traditional Chinese medicine). Gamboge possesses anti-inflammatory, anti-viral and anti-cancer effects (Xu and Yang, 2013; Yen et al., 2012; Wen et al., 2014; Kong et al., 1996). As a promising natural anti-cancer candidate, GA’s activity in numerous types of cancer cells has been well demonstrated in vivo and in vitro, including leukemia, lung, liver, SH-SY5Y neuroblastoma, breast, colorectal, U87 glioma, ovarian and gastrointestinal cancer cells (Chen et al., 2014; Zhang et al., 2014; Qi et al., 2014; Duan et al., 2014; He et al., 2013; Wang and Yuan, 2013; Rahman et al., 2013; Li et al., 2012; Zou et al., 2012). In addition to significant anti-tumor effects, GA also exhibits anti-inflammatory, detoxification and parasiticide effects (Yen et al., 2012; Wen et al., 2014; Rahman et al., 2013). However, although GA shows various pharmacological activities, information regarding the in vivo fate of GA is still limited. There has been no report for GA bioavailability, excretion or tissue distribution with a sensitive method either in humans or in animals. To date, several techniques have been employed for analysis of GA in Gamboge (only in the crude herb, not in biomatrices) for quality assessment, such as high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection (Song et al., 2007; Li et al., 2008), capillary electrophoresis with UV detection (Lopez-Montes et al., 2007) and HPLC with evaporative light-scattering detection (Yang et al., 1999). HPLC tandem electrospray ionization mass spectrometry (HPLC-ESI/MS) has
Biomed. Chromatogr. 2015
been utilized for the identification of xanthones in Gamboge herb (Zhou et al., 2008), and for structural identification for GA metabolites in rats or human (Feng et al., 2007; Yang et al., 2010). A HPLC-UV method has been reported to study pharmacokinetics in dog plasma with a lowest limit of quantification (LLOQ) of 156 ng/mL (Hao et al., 2007a, 2007b). Ding et al. (2007) developed a liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry with single ion monitoring method for the determination of GA in human plasma with LLOQ of 31 ng/mL. In above three reports the administration route was via intravenous injection. These methods might be sensitive enough for the quantification of GA in biosamples after intravenous injection. Recently, Hua et al. (2014) quantified GA and gambogenic acid in rat plasma after oral administration of Garcinia hanburyi extracts by LC-MS. Bioavailability (BA), excretion and tissue distribution for GA were not reported in the above paper.
* Correspondence to: Y. Li, School of Life Science, Beijing Institute of Technology, Beijing 100081, China. Email: [email protected] a
School of Life Science, Beijing Institute of Technology, Beijing 100081, China
b
Hebei North University, Zhangjiakou 075000, China
c
China Astronaut Research and Training Centre, Beijing 100094, China Abbreviations used: BA, bioavailability; GA, gambogic acid; MRM, multiple reaction monitoring.
Copyright © 2015 John Wiley & Sons, Ltd.
Z. Zheng et al. generation of precursor and fragment ions. Optimum parameters were as follows: nebulizer (GS1), heater (GS2) and curtain gas flow rates, 40, 50 and 10 arbitrary units, respectively; heater gas temperature, 500 °C; ion spray needle voltage, 4500 V; declustering potential, 60 and 50 V for GA and IS, respectively; collision energies, 23 and 28 V for GA and IS, respectively; collision potential, 11 and 8 V for GA and IS, respectively; cell exit potential, 18 and 10 V for GA and IS, respectively.
Figure 1. Chemical structures of gambogic acid (GA) (A) and IS (IS).
It is known that different bio-matrices need different fully validated methods. To our knowledge, HPLC-MS methods are not available for the determination of GA in urine, bile or tissues of experimental animals or human. Few studies have been carried out for evaluating urinary and biliary excretion or tissue distribution of GA in rats with HPLC-MS. In addition, reports on the dosedependent pharmacokinetics of single GA in rats after oral administration have been not proposed elsewhere. Further, the oral bioavailability of GA was not previously available. Therefore, the present study aimed to develop a reliable and sensitive ultra-HPLC-MS method with an LLOQ of 1.0 ng/mL for quantification of GA in rat plasma, urine, bile and main tissues. It has been applied to the investigation of BA, pharmacokinetics, excretion and tissue distribution in rats for the first time. The expected results might supply more in vivo information for further clinical application of GA.
Calibration standards and quality control sample preparation A 1.0 mg aliquot of GA was dissolved in 1.0 mL of methanol to give the concentration of 1.0 mg/mL as the stock solution. Working solutions were prepared by dilution of the stock solution with methanol–water (9:1, v/v, 90% methanol) to obtain series of working concentrations. All of the solutions were stored at 4 °C before use. A 1.0 mg aliquot of phenytoin sodium (IS) was dissolved in 1.0 mL of methanol to give the concentration of 1.0 mg/ mL as the IS stock solution. IS solution was finally diluted to 20 ng/mL with acetonitrile and kept at 4 °C until use. The calibration standards (1, 2, 5, 10, 50, 100, 500 and 1000 ng/mL) were prepared by spiking blank plasma or bile with appropriate amounts of working solutions to yield a final concentration range of 1.00–1000 ng/ mL. For urine and tissue calibration curves, calibration standards (1, 2, 5, 10, 50, 100 and 500 ng/mL) were prepared by spiking blank urine or tissues with appropriate amounts of working solutions to yield a final concentration range of 1.00–500 ng/mL. Quality control (QC) samples were prepared with at three concentration levels of 1, 80 and 800 ng/mL for rat plasma and bile, and 1, 80 and 400 ng/mL for rat urine and tissues.
Sample preparation
Experimental Chemicals and reagents GA (99.1%, purity) was supplied by Shanghai Yuanye Biotech Company (Shanghai, China). Phenytoin sodium (98.9%, purity, used as internal standard, IS) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The chemical structures of GA and IS are shown in Fig. 1. Methanol and acetonitrile were of chromatographic grade and supplied by Thermo Fisher Scientific (California, USA). Ammonium acetate was of analytical grade from Yili Company (Beijing, China). Distilled water, prepared using a Milli-Q water Millipore purification system (Molsheim, France), was used throughout the study.
A 50 μL aliquot of plasma samples (or blank plasma) was mixed with 5 μL of 90% methanol (or working solutions) and 150 μL of IS solution in vials. The mixture was vortexed for 2 min. The samples were centrifuged at 10,000 g for 10 min. A 10 μL aliquot of supernatant was injected onto the chromatographic column for analysis. For urine, bile and tissue homogenates samples, 10 μL of samples (or blank urine, bile, tissue homogenate) was transferred to vials and mixed with 5 μL of 90% methanol (or working solutions). After 150 μL of IS solution was added, the mixture was thoroughly vortexed for 2 min. The samples were centrifuged at 10,000 g for 10 min. A 10 μL aliquot of supernatant was injected onto the chromatographic column for analysis.
Method validation LC-MS/MS conditions The instrument used in the present experiment mainly contained an Agilent 1290 UHPLC system (Agilent Technologies, Mountain View, California, USA), TM an Applied Biosystems Sciex 4000 Q-trap mass spectrometer via a Turbo Ion Spray ionization interface (Concord, Ontario, Canada) and an Infinity sampler G4226A (Agilent, California, USA). Applied Biosystems Analyst software version 1.5.1 package (Applied Biosystems, California, USA) was used to control the UHPLC-MS system and for data acquisition and processing. An Agilent Zorbax XDB-C18 column (50 × 2.1 mm, 1.8 μm, Agilent Technologies, California, USA) was used for chromatographic separation. The mobile phase consisted of a mixture of 0.06% ammonium acetate in water (A) and 0.06% ammonium acetate in acetonitrile–methanol (1:1, v/v) (B). The gradient elution was programmed as follows: 0.0–0.4 min 80% A; 0.4–1.0 min 20% A; 1–4.0 min, 5% A; 4.0–5.0 min, 80% A. The flow rate was 0.2 mL/min and the column temperature was set at 25 °C. The mass spectrometer was operated in the negative electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) at unit resolution. Nitrogen was used as the nebulizer, heater and curtain gas as well as the collision-activated dissociation gas. The precursor-to-product ion transitions were monitored at m/z 627.3 → 583.2 for GA and m/z 251.2 → 101.9 for IS. Mass spectrometer parameters were tuned to maximize the
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The method was validated according to the Food and Drug Administration guidance for bioanalytical method validation, including linearity, selectivity, precision and accuracy, recovery and stability (US Food and Drug Administration, 2001). The matrix effect was also evaluated following the published method (Matuszewski et al., 2005). 2 Linearity was assessed by weighted linear regression (1/x , where x is the concentration of GA in rat plasma, bile, urine or tissues) of analyte–internal standard peak area ratios (y) based on three independent calibration curves prepared on each of three separate days. Selectivity was tested by comparison of blank matrices from six individual rats with corresponding spiked matrices. At the same time, spiked solutions with calibration concentration levels were used to determine calibration curve. QC samples at three concentrations in six replicates were analyzed during the same day using the same calibration curve to determine the intra-day precision. All of the samples mentioned above were analyzed for three continuous days to define inter-day precision. Precision was expressed as the relative standard deviation (RSD). Accuracy was calculated as the relative error (RE). The lowest limit of quantitation (LLOQ) is the lowest concentration of analyte on the calibration curve, which should be quantified reliably with an acceptable accuracy and precision (≤15 %; US Food and Drug Administration, 2001).
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Biomed. Chromatogr. 2015
Bioavailability, excretion, distribution of gambogic acid
Figure 2. Typical multiple reaction monitoring (MRM) chromatograms of (A) blank plasma; (B) blank plasma spiked with GA (10 ng/mL) and IS (20 ng/mL); (C) a real rat plasma sample at 4 h post-dosing; (D) a real rat bile sample within 0–4 h post-dosing; (E) a real rat urine sample within 12–24 h post-dosing; (F) a real rat liver sample at 1 h post-dosing. Peak I, GA; peak II, IS.
Table 1. Calibration range, equation, correlation coefficient and lowest limit of quantification (LLOQ) of gambogic acid (GA) in all biological matrices Matrix
Calibration range (ng/mL)
Calibration equation
Correlation coefficient (r)
LLOQ (ng/mL)
Plasma Bile Urine Heart Liver Spleen Lung Kidney Brain
1.00–1000 1.00–1000 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500 1.00–500
y = 0.429x + 0.032 y = 0.043x + 0.046 y = 0.030x + 0.027 y = 0.031x + 0.029 y = 0.024x + 0.018 y = 0.018x + 0.062 y = 0.048x + 0.026 y = 0.023x + 0.031 y = 0.003x + 0.022
0.9959 0.9964 0.9961 0.9952 0.9949 0.9961 0.9980 0.9979 0.9980
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
Table 2. Precision and accuracy for determination of GA in rat plasma, urine, bile and liver by UHPLC-MS Method (n = 6) Matrix
Concentration ng/mL
Plasma
Bile
Urine
liver
1.00 80.0 800.0 1.00 80.0 800.0 1.00 80.0 400.0 1.00 80.0 400.0
Precision (RSD, %)
Accuracy (RE, %)
Intra-day
Inter-day
Intra-day
Inter-day
3.4 2.8 7.1 2.7 9.2 2.6 4.6 7.4 1.4 6.6 4.8 3.6
9.0 1.7 1.8 2.8 3.4 8.7 9.1 2.8 3.3 4.9 1.6 2.7
6.8 –2.1 3.2 2.4 8.6 –1.9 –4.7 8.8 –5.6 4.7 –2.1 –0.9
–3.9 –3.4 6.8 7.7 –5.6 8.9 3.2 4.6 –6.7 2.1 –3.4 7/9
Note, accuracy and precision for other tissues are not listed here.
Biomed. Chromatogr. 2015
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2.2
–2.9 3.4
–8.1 –1.7
–0.8
6.2
2.9
5.1
–0.9 2.1 3.2
1.8 4.5
2.6 –7.7
Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed urine samples at 25 °C (RE %) 8h Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed liver samples at 25°C (RE %) 8h 2.6 –3.1 –5.8
–4.9 2.6 3.4
2.1 –2.6
2.6 –6.7
3.4
2.9 –3.1
1.9
Liver
Urine 2.8 –6.4 1.6
800 80.0 1.00
–8.1
Conditions Matrix Spiked concentration (ng/mL)
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Note, stabilities for other tissues are not listed here.
The rats were put into metabolism units to collect urine samples at 4, 8, 12, 24, 36, 48 and 60 h after dosing. Rats for biliary excretion experiment were first anesthetized with 3 g/kg of chloral hydrate and were surgically implanted with a cannula in the bile duct. Bile samples were collected at 4, 8, 12 and 24 h post-ingestion. Blank urine and bile was collected before dosing. The volumes of urine and bile samples were recorded. The urine and bile samples were centrifuged at 10,000 g for 10 min and then all supernatant was kept at 20 °C until use.
Bile
Excretion after oral administration
Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed plasma samples at 25 °C (RE %) 8h Freeze–thaw stability (RE %) 3 cycles Storage stability at 20°C (RE %) 30 days Processed bile samples at 25°C (RE %) 8h
In tissue distribution experiment, rats (n = 6 for each time point) were fasted for 12 h with free access to water. After rats were orally administered 80 mg/kg GA, main tissues (heart, liver, spleen, lung, kidney and brain) were collected at 0.5, 1 and 4 h post-dosing and then weighed. Blank tissues samples were collected separately before ingestion. Tissues were homogenized with saline (2-fold the tissue weight) and then centrifuged at 10,000 g for 10 min to get the homogenates. The homogenate samples were stored at –20 °C until use.
Plasma
Tissue distribution after oral administration
Conditions
For two oral administration (p.o.) groups (n = 6 for each group), all animals were fasted for 12 h prior to administration with free access to water. GA was suspended in 5% (w/v) carboxy-mehtylcellulose sodium (CMC-Na) solution, and then was administrated by gastric gavage (40 and 80 mg/kg to rats, respectively). In intravenous administration (i.v.) groups (n = 6 for each group), GA dissolved in saline (containing 0.4% Tween 80 and 6% PEG300) was given via the caudal vein (4 mg/kg to rats). A blood sample (0.2 mL) was collected from orbital vein into heparinized tubes at appropriate intervals (10, 20, 30, 45 min, 1, 2, 4, 6, 9 and 12 h for p.o. group; 2, 5, 10, 30 min, 1, 2, 3, 4, 6 and 9 h for i.v. group) after dosing. A blank plasma sample was collected before dosing. All the blood samples were centrifuged at 10,000 g for 10 min and then plasma samples were stored at –20 °C until analysis.
Matrix
Oral bioavailability and pharmacokinetics
Table 3. Stability of GA in rat plasma and urine under different storage conditions determined by UHPLC-MS method (n = 6)
Animal treatment and sample collection Sprague–Dawley rats (male, 200–220 g body weight) were purchased from Beijing Military Medical Sciences Experimental Animal Co. Ltd (Beijing, China). All the rats were housed in an air-conditioned room with the temperature of 24 ± 2 °C, relative humidity of 55 ± 10% and alternating 12 h light/dark cycle. The study complied with guidelines for the Care and Use of Laboratory Animals (published by the National Institutes of Health, NIH publication no. 85-23). The experimental procedures were approved by Beijing Institute of Technology Committee on Animal Care and Use [Beijing, China. Permission Number: SYXK ( Jing) 2012-0035].
2.6
400 80.0 1.00
Spiked concentration (ng/mL)
The recovery of analytes and IS was determined by comparing the peak areas of the analytes and IS in deproteinized QC samples with those in post-deproteinization blank samples spiked at the corresponding concentrations. Freeze–thaw stability of GA in rat plasma, urine, bile and tissues was assessed using four concentration of QC samples after three freeze (–20 °C) to thaw (25 °C) cycles. Long-term stability of QC samples was evaluated after storage at –20 °C for 30 days. The stability of the analyte in the all matrices was also investigated at 25 °C for 8 h. The sample was considered as stable if the relative error was 100% mean ionization enhancement, whereas ME values < 100% indicate ionization suppression.
3.4
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Biomed. Chromatogr. 2015
Bioavailability, excretion, distribution of gambogic acid 150
Methanol was first considered as elution solvent. However methanol showed poor peak shape for GA. The mixture of methanol and acetonitrile was proved to achieve proper running time and to be more economical than acetonitrile itself. After thorough optimization, the gradient elution procedure was finally chosen because it led to better peak symmetry and shorter retention times for GA and IS than isocratic elution. The retention times of GA and IS under the chromatographic conditions used were 3.58 and 2.13 min, respectively. IS was indispensable for the bio-analysis. Several internal standards, such as loureirin B, spinosin and phenytoin sodium, were tested in the present study. Spinosin showed stronger ion responses in positive than negative ion mode. Loureirin B could give a good response in both modes, while it did not give a satisfactory retention time (0.9949. The calibration equation, calibration range, LLOQ and correlation coefficient (r) in all matrices of GA are listed in Table 1. Intra-day and inter-day precisions (RSD) and accuracy (RE) for quantification of GA in rat plasma, urine, bile and liver are summarized in Table 2. Accuracy for plasma samples ranged from –6.7 to 8.8 % RE. For bile samples, accuracy was between –1.9 and 8.6 %
A
40 mg/kg,po 80 mg/kg,po
120 90 60 30 0
2
0
4
6
8
10
12
Time (h) 1000
Concentration (ng/mL)
Method development
Concentration (ng/mL)
Results and discussion
B
4 mg/kg,iv
800 600 400 200 0 0
2
4
6
8
10
Time (h) Figure 3. Mean plasma concentration–time curve of GA after oral administration to rats. (A) 40 and 80 mg/kg p.o.; (B) 4 mg/kg i.v. Data is expressed as mean ± SD (n = 6 for each time point).
RE. Accuracy for urine and liver was in the range of –5.6–8.8% RE. The intra- and inter-day precisions for plasma, urine, and bile and liver were
