Research article Received: 6 December 2013,
Revised: 15 March 2014,
Accepted: 31 March 2014
Published online in Wiley Online Library: 10 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3225
Quantification of homoegonol in rat plasma using liquid chromatography–tandem mass spectrometry and its pharmacokinetics application Hyeon-Uk Jeonga†, Soon Sang Kwona†, Deok-Gyu Hwanga, Eun Nam Kima, Kyeong Leeb, Kyung-Seop Ahnc, Sei-Ryang Ohc and Hye Suk Leea* ABSTRACT: Homoegonol is a biologically active neolignan isolated from Styrax species with cytotoxic, antimicrobial, antiinflammatory and anti-asthma activities. For the quantification of homoegonol in rat plasma, a selective and sensitive liquid chromatography–tandem mass spectrometric method was developed and validated for the first time using protein precipitation with methanol as a sample clean-up procedure. The analytes were separated in an Atlantis dC18 column using a gradient elution of methanol and 0.1% formic acid, and mass-to-charge ratios were determined in selective reaction monitoring mode using tandem mass spectrometry with m/z 343.12 > 296.97 for homoegonol and m/z 517.30 > 282.90 for udenafil (internal standard). The standard curve was linear over the concentration ranges of 1 500 ng/mL using a 30 μL rat plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four quality control levels were 3.9–10.0 and -3.3–2.7%, respectively. The overall recovery of homoegonol from rat plasma using protein precipitation was 99.7 ± 7.7%. The pharmacokinetics parameters of homoegonol were dose-independent after both intravenous (1, 2.5 and 5 mg/kg doses) and oral (5, 10 and 20 mg/kg doses) administration in male Sprague–Dawley rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: homoegonol; liquid chromatography-tandem mass spectrometry; rat plasma; pharmacokinetics
Introduction
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Homoegonol is a biologically active neolignan isolated from Styrax species, such as Styrax camporum, Styrax japonica and Styrax pohlii (Bertanha et al., 2012; Moraes et al., 2012; Teles et al., 2005) and showed cytotoxic (Hirano et al., 1994; Teles et al., 2005), antimicrobial (Ozturk et al., 2008; Pauletti et al., 2000), anti-inflammatory (Bertanha et al., 2012; Moraes et al., 2012; Rodrigues and de Carvalho, 2008) and anti-asthmatic effects in an ovalbumin-induced murine asthma model (Shin et al., 2014). Asthma is a complex chronic inflammatory airway disease with substantial increase in incidence. Numerous drugs have been used for treating asthma, but the use of drugs is limited owing to low efficacy or side effects (Durham et al., 2011). New drug development for the treatment of asthma is urgently needed. Based on our previous results (Shin et al., 2014), homoegonol is currently being evaluated in various efficacy and toxicity studies for development of anti-asthmatic drugs. The evaluation of the bioavailability and pharmacokinetics of homoegonol can link the data from pharmacological assays to clinical effects and design the rational dosage regimens. However, there has been no report on the pharmacokinetics of homoegonol or a bioanalytical method for the quantification of homoegonol in biological fluids. The purpose of the present paper was to develop a rapid, reproducible, selective and sensitive LC-tandem mass spectrometric (LC-MS/MS) method using electrospray ionization for quantification of homoegonol in rat plasma and to characterize
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the pharmacokinetics of homoegonol after intravenous and oral administration at various doses in male Sprague–Dawley rats.
Experimental Materials and reagents Homoegonol was obtained from Toronto Research Chemicals Inc. (Toronto, Canada). Udenafil (used as an internal standard, IS) was a gift from Dong-A Pharmaceutical Co. (Yongin, Korea). Methanol and water (LC-MS grade) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Other chemicals used were of the highest quality available. Drug-free rat
* Correspondence to: Hye Suk Lee, Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea. Email: [email protected] †
The first two authors contributed equally.
a
Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
b
College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea
c
Natural Medicine Research Center, Korea Research Institute of Biology and Biotechnology, Chungbuk 363-883, Republic of Korea Abbreviations used: SRM, selected reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
LC-MS/MS of homoegonol in rat plasma plasma containing sodium heparin as the anticoagulant was prepared from male Sprague–Dawley rats.
Preparation of calibration standards and quality control samples Stock solutions of homoegonol (1 mg/mL) and udenafil (1 mg/mL) were prepared in dimethyl sulfoxide. The stock solution of homoegonol was serially diluted with acetonitrile to working standard solutions. Udenafil stock solution was diluted to 5 ng/mL with methanol. All standard solutions were stored at 4 °C for 4 weeks in amber glass vials in the dark when not in use. Rat plasma calibration standards of homoegonol at concentrations of 1, 2, 5, 10, 50, 200, 400 and 500 ng/mL were prepared by addition of 1.5 μL of the working standard solutions (0.02, 0.04, 0.1, 0.2, 1, 4, 8 and 10 μg/mL) to 30 μL of drug-free rat plasma. To prepare quality control (QC) samples at 1, 3, 30, 375 and 7500 ng/mL, 60 μL of the appropriate working standard solutions at 0.1, 0.3, 3, 37.5 and 750 μg/mL were added to 5940 μL of drug-free rat plasma. Aliquots (30 μL) of QC samples were transferred into polypropylene tubes and stored at 80 °C until analysis.
Sample preparation
Pharmacokinetic study of homoegonol in rats
A 30 μL aliquot of rat blank plasma, calibration standards and QC samples were vortex-mixed with 100 μL of udenafil in methanol (5 ng/mL, IS) for 3 min at high speed. After centrifugation at 13,000 rpm at 4 °C for 8 min, 50 μL of the supernatant was diluted with 50 μL of water. The aliquot (7 μL) was injected into the LC-MS/MS.
LC-MS/MS analysis The LC-MS/MS system consisted of a Nanospace SI-2 nano-LC (Shiseido, Tokyo, Japan) coupled with a Finnigan Quantum Access tandem mass spectrometer (Thermo Scientific, San Jose, CA, USA). Separation was performed on the Atlantis dC18 column (5 μm, 2.1 mm i.d. × 100 mm, Waters Co.) using gradient elution of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.3 mL/min: 40% mobile phase B for 0.5 min, 40% to 95% mobile phase B for 0.1 min, 95% mobile phase B for 4.9 min, 95–40% mobile phase B for 0.1 min, 40% mobile phase B for 4.4 min. The column and autosampler were maintained at 50 and 6 °C, respectively. The electrospray ionization source settings for analysis of homoegonol and udenafil were as follows: spray voltage, 4.5 kV; vaporizer temperature, 350 °C; capillary temperature, 330 °C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 15 psi. The tube lens offsets for homoegonol and udenafil were 60 and 94 V, respectively. Fragmentation + of the [M + H] ion for homoegonol and udenafil was performed at a collision energy of 19 and 40 V, respectively, by collision-activated dissociation with argon gas as the collision gas at a pressure setting of 1.3 on the instrument. Selected reaction monitoring (SRM) mode was employed for the quantification: m/z 343.12 → 296.97 for homoegonol and m/z 517.30 → 282.90 for udenafil. Xcalibur® software (ThermoFisher Scientific) was used for the LC-MS/MS system control and data processing.
Method validation
This method was applied to the pharmacokinetic study of homoegonol after intravenous and oral administration at various doses in male Sprague–Dawley rats (body weight 229 263 g) (Samtako Co., Osan, Korea). The animal experiments were approved by institutional Animal Ethical Committee. Animals were kept in plastic cages with free access to standard rat diet (Samtako Co.) and water before the experiment. Animals were maintained at a temperature of 22 24 °C with a 12 h light/dark cycle and relative humidity of 50 ± 10%. Rats were anesthetized by isoflurane and were cannulated with polyethylene tubing (PE-50, Natsume Co., Tokyo, Japan) in the jugular vein for blood sampling and in the femoral vein for intravenous injection. Each rat was housed individually in a rat metabolic cage and allowed to recover from anesthesia for 1 day prior to the start of the study. Rats were not restrained at any time during the study. In order to prevent blood clotting, each catheter was flushed with heparin in physiological saline solution (10 U/mL). Homoegonol was dissolved in a mixture of N,N-dimethylacetamide, Tween 80 and saline (6:1:8, v/v) and was administered to rats by bolus injection via the femoral vein at 1 (n = 3), 2.5 (n = 4) and 5 (n = 4) mg/kg doses. For the oral study, homoegonol (the same solution used in the intravenous study) was administered to rats by stomach gavage tube at doses of 5 (n = 4), 10 (n = 7) and 20 mg/kg (n = 5). Blood samples (150 μL) were collected before (control) and at the following time points following drug administration: 2 (only for intravenous), 5, 15, 30, 45, 60 min, and at 1.5, 2, 3, 4, 6, 8, 12 and 24 h. Plasma samples were harvested by centrifugation at 13,000 rpm for 8 min and stored at 80 °C until analysis. The pharmacokinetic parameters of homoegonol were analyzed by a noncompartment analysis (WinNonlin, Pharsight, Mountain View, CA, USA): the total area under the plasma concentration–time curve (AUC), the terminal half-life (t1/2), the time-averaged total body, renal and nonrenal clearances (Cl, Clr, and Clnr, respectively), and the apparent volume of distribution at steady-state (Vss). The peak plasma concentration (Cmax) and the time to reach Cmax (Tmax) were directly obtained from the experimental data. The bioavailability (F) values were calculated by dividing dose-normalized (at 2.5 mg/kg) AUC obtained after oral administration by the AUC after the intravenous administration at a dose of 2.5 mg/kg. All data are expressed as the means ± SD.
Results and discussion LC-MS/MS Electrospray ionization of homoegonol and udenafil produced abundant [M + H]+ ions at m/z 343.12 and 517.30, respectively,
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In order to complete the method validation, the batches consisting of triplicate calibration standards covering the range of 1-500 ng/mL and five replicates of QC samples at 1, 3, 30, and 375 ng/mL were analyzed on three consecutive days. The relative error (RE), the percentage of deviation of the mean from the true values, served as a measure of the accuracy, and the coefficient of variation (CV) indicated the precision. To demonstrate selectivity, rat plasma from 10 different rats was screened for interference at the retention time and mass transition of homoegonol and udenafil (IS). The matrix effect for homoegonol and udenafil (IS) was investigated according to the bioanalysis method validation guideline of the
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European Medicines Agency. The matrix factor was evaluated by calculating the ratio of the peak area of analyte spiked after extraction into plasma extracts originating from six different rats to the peak areas for the pure solution of the analyte in 40% methanol at 1, 3 and 375 ng/mL. The IS-normalized matrix factor was calculated by dividing the matrix factor of homoegonol by the matrix factor of the IS. The CV of the IS-normalized matrix factor from the six different rat plasmas should not be greater than 15%. Recovery of homoegonol and udenafil was determined by comparing the mean peak areas of the analytes spiked before extraction into the blank plasma sources with those of the analytes spiked post-extraction into the blank plasma extracts at four concentrations, which were 1, 3, 30 and 375 ng/mL. QC samples at 7500 ng/mL were diluted 20-fold with blank rat plasma to establish the concentration above the upper limit of the calibration curve that can be diluted into quantifiable range. For evaluation of freeze–thaw stability, long-term storage stability, short-term storage stability and room temperature storage stability, five replicates of the QC samples at low and high concentrations (3 and 375 ng/mL) were subjected to three freeze–thaw cycles, storage at 80 °C for 25 days, or storage at room temperature for 2 h before processing. Post-extraction batch integrity was determined by batch reinjection after 24 h of storage in the autosampler.
H.-U. Jeong et al. without evidence of fragmentation or adduct formation. The [M + H]+ ion of homoegonol and udenafil was selected as the precursor ion and was subsequently fragmented in MS/MS mode in order to obtain the product ion spectra, yielding useful structural information (Fig. 1). Homoegonol produced the major product ion at m/z 296.97 by loss of ethanol from the [M + H]+ ion, and udenafil showed a prominent product ion at m/z 282.90 [5-(2-hydroxy-phenyl)-1-methyl-3-propyl-1,4-dihydropyrazolo[4,3-d]pyrimidin-7-one radical]. For the quantification of homoegonol in the plasma, SRM mode was used owing to the high selectivity and sensitivity of SRM data acquisitions, where the transition of the precursor ion to a product ion was monitored as follows: m/z 343.12 to m/z 296.97 for homoegonol and m/z 517.30 to m/z 282.90 for udenafil. Homoegonol showed better retention and peak shape on an Atlantis dC18 column compared with a Pinnacle biphenyl column and octadecyl-bonded phase columns such as Imtakt C18 and Halo C18. Use of methanol as an organic solvent in the mobile phase increased the organic solvent content compared with acetonitrile and resulted in high sensitivity of homoegonol owing to the increase of electrospray ionization efficiency.
Method validation Calibration curves were obtained over the concentration ranges of 1–500 ng/mL of homoegonol in rat plasma. Linear regression analysis with a weighting of 1/concentration gave the optimum accuracy (RE, 2.0–5.1%) and precision (CV, ≤13.2%) of the corresponding calculated concentrations at each level (Table 1). The low CV value (7.4%) for the slope indicated the repeatability of the method (Table 1). Table 2 shows a summary of the intra- and inter-day precision and accuracy data for QC samples containing homoegonol. Both intra- and inter-day CV values ranged from 3.9 to 10.0% at four QC levels. Intra- and inter-assay RE values were 3.3 to 2.7% at four QC levels. These results indicated acceptable accuracy and precision of the present method. The lower limit of quantification (LLOQ) for homoegonol was set at 1 ng/mL using 30 μL of rat plasma with a signal-to-noise ratio >10 (Fig. 2b). After a 20-fold dilution of the dilution quality control samples at 7500 ng/mL, the CV and RE for homoegonol were 5.0 and 5.8%, respectively, indicating the acceptability of the 20-fold dilution prior to analysis. In the analysis of the blank plasma samples obtained from 10 different rats, no interference peak was observed at the retention times of homoegonol (3.63 min) and udenafil (3.03 min), indicating the selectivity of the present method (Fig. 2a). Sample carryover effect was not observed. The matrix factor for homoegonol at 1, 3 and 375 ng/mL was 0.96 ± 0.09, 0.94 ± 0.06 and 0.96 ± 0.03, respectively. The matrix factor for IS was 0.91 ± 0.06. The IS-normalized matrix factor for homoegonol at 1, 3, and 375 ng/mL was 1.08 ± 0.06, 1.09 ± 0.06, and 1.01 ± 0.05, respectively. The CV values of the IS-normalized matrix factor from the six different rat plasmas were 5.9, 5.2 and 4.9% for 1, 3 and 375 ng/mL, respectively. These data confirm that the matrix effects for homoegonol and udenafil have little effect on the determination of homoegonol.
Table 2. Precision and accuracy of homoegonol in quality control (QC) samples Statistical variable
Figure 1. Product ion mass spectra of (a) homoegonol and (b) udenafil (internal standard).
Intra-day (n = 5)
Inter-day (n = 3)
QC (ng/mL) 1.0 3.0 30.0 375.0 1.0 3.0 30.0 375.0 Mean 1.0 2.9 30.8 368.5 1.0 2.9 30.3 376.8 (ng/mL) CV (%) 10.0 4.7 5.2 5.2 9.4 7.8 4.3 3.9 RE (%) 0.0 –3.3 2.7 –1.7 0.0 –3.3 1.0 0.5
Table 1. Calculated concentrations of homoegonol in calibration standards prepared with rat plasma (n = 9) Statistical variable Mean (ng/mL) CV (%) RE (%)
Theoretical concentration (ng/mL)
Slope
1.0
2.0
5.0
10.0
50.0
200.0
400.0
500.0
1.0 13.2 0.0
2.0 2.0 0.0
4.9 9.6 –2.0
9.8 4.1 –2.0
50.9 1.5 1.8
210.2 1.0 5.1
392.0 3.1 –2.0
497.3 2.7 –0.5
0.0889 7.4
Intercept
0.0094
r2
0.9983 0.1
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CV, Coefficient of variation; RE, relative error
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LC-MS/MS of homoegonol in rat plasma
Figure 2. Selected reaction monitoring chromatograms of (a) a rat blank plasma, (b) a rat plasma sample spiked with 1 ng/mL of homoegonol, and (c) a rat plasma sample obtained 15 min after intravenous administration of homoegonol at a dose of 2.5 mg/kg to a male Sprague–Dawley rat.
The overall recovery of homoegonol from rat plasma using protein precipitation was 99.7 ± 7.7% for four QC concentration levels and was consistent over the concentration range of 1–375 ng/mL. Recovery of the internal standard udenafil was 91.4 ± 3.8%. The stability of processing (freeze–thaw, long-term storage at 80 °C, and short-term storage at room temperature) and chromatography (re-injection) was assessed and was shown to be of insignificant effect (Table 3). Three freeze–thaw cycles, long-term storage at 80 °C for 25 days, and room temperature storage of QC samples for 2 h at low and high concentrations prior to analysis had little effect on quantification. Re-analysis of the organic extracts stored for 24 h at 6 °C showed acceptable accuracy and precision of the QC samples. Pharmacokinetics of homoegonol in rats The method was applied successfully to study pharmacokinetics after intravenous injection of homoegonol at 1, 2.5 and 5 mg/kg doses and oral administration of homoegonol at 5, 10 and
Table 3. Stability of quality control samples (n = 5) Statistical variable
20 mg/kg doses in male Sprague–Dawley rats. Figure 2(c) shows representative SRM chromatograms from the analysis of a plasma sample obtained 15 min after intravenous administration of homoegonol at 2.5 mg/kg dose in a rat. After intravenous administration of homoegonol, its mean plasma concentration–time curves declined in a poly-exponential fashion for all three doses studied (Fig. 3). Its AUCs were linearly increased as the dose increased (Table 4). The dose-normalized (based on 1 mg/kg) AUCs were comparable among three doses studied: the values were 126.4 ± 9.8, 152.6 ± 32.5 and 132.6 ± 38.2 ng h/mL for 1, 2.5, and 5 mg/kg, respectively. Moreover, the slope between the log AUC and log dose (1.0388) was close to 1. The other pharmacokinetic parameters of homoegonol shown in Table 4 were also comparable among three doses studied, indicating that the pharmacokinetic parameters of intravenous homoegonol were independent of dose. The contribution of the renal clearance to the clearance of homoegonol was negligible: Ae0–24h was 0.001–0.006% for all three doses studied. This indicated that homoegonol is almost completely eliminated via the nonrenal route. After oral administration, homoegonol was rapidly absorbed and was detected at the first blood sampling time point (5 min) with rapid Tmax, 0.22–0.50 h for all three doses (Fig. 4, Table 5). As the oral dose increased, AUC and Cmax were linearly increased
Theoretical concentration (ng/mL) 3.0
375.0
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Figure 3. Mean plasma concentration–time profiles of homoegonol after its intravenous administration at 1 (●, n = 3), 2.5 (▼, n = 4) and 5 (■, n = 4) mg/kg to rats. Bars represent SD.
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Three freeze–thaw cycles Mean (ng/mL) 2.8 371.7 CV (%) 3.4 9.4 RE (%) –6.7 –0.9 Short-term stability (2 h storage at room temperature) Mean (ng/mL) 3.2 365.3 CV (%) 4.7 2.4 RE (%) 6.7 –2.6 Long-term storage stability (25 days at –80 °C) Mean (ng/mL) 2.7 343.6 CV (%) 4.9 1.6 RE (%) –10.0 –8.4 Post-preparative stability (24 h in autosampler) Mean (ng/mL) 3.0 390.9 CV (%) 7.6 2.1 RE (%) 0.0 4.2
H.-U. Jeong et al. Table 4. Mean pharmacokinetic parameters of homoegonol after its intravenous administration at various doses to male rats Parameter
1 mg/kg (n = 3) a
AUC (ng h/mL) Cl (mL/min/kg) Clr (mL/min/kg) Clnr (mL/min/kg) Vss (L/kg) t1/2 (h) Ae0–24h (percentage of the dose)
126.4 ± 9.8 130.25 ± 9.63 0.006 ± 0.005 130.25 ± 9.63 3.11 ± 0.43 (0.49 ± 0.13)b 0.005 ± 0.004
2.5 mg/kg (n = 4)
5 mg/kg (n = 4)
381.4 ± 81.3 112.69 ± 25.35 0.007 ± 0.002 112.69 ± 25.35 2.92 ± 0.37 0.88 ± 0.20 0.006 ± 0.001
662.8 ± 191.1 133.02 ± 36.92 0.002 ± 0.003 133.02 ± 36.92 4.84 ± 1.52 1.29 ± 0.38 0.001 ± 0.002
a
Dose-normalized (1 mg/kg) AUCs were compared for statistical analysis. The numbers in parentheses represent pharmacokinetic parameters of homoegonol at the dose of 1 mg/kg. Because the detection of plasma concentrations of homoegonol at 1 mg/kg was shorter than those at 2.5 and 5 mg/kg, these data were not included in statistical analysis. AUC, total area under the plasma concentration-time curve; t1/2, the terminal half-life; Cl, Clr, and Clnr, the time-averaged total body, renal and nonrenal clearances, respectively; Vss, the apparent volume of distribution at steady-state; Ae0–24h, the percentage of the dose of homoegonol excreted in 24 h urine. b
Table 5. Mean pharmacokinetic parameters of homoegonol after its oral administration at various doses to rats Parameter AUC (ng h/mL)a Tmax (h) Cmax (ng/mL)a t1/2 (h) Ae0–24h (percentage of the dose) F (%)
5 mg/kg (n = 4)
10 mg/kg (n = 7)
20 mg/kg (n = 5)
6.88 ± 5.54 0.50 ± 0.20 9.1 ± 5.3 (0.50 ± 0.26)b NDc 0.90 ± 0.73
16.37 ± 9.54 0.50 ± 0.20 19.2 ± 6.0 0.49 ± 0.16 0.003 ± 0.003 1.07 ± 0.62
22.67 ± 18.23 0.22 ± 0.07 27.1 ± 17.3 0.69 ± 0.28 0.004 ± 0.008 0.74 ± 0.60
a
Dose-normalized (5 mg/kg) AUCs and Cmax were compared for statistical analysis. The numbers in parentheses represent pharmacokinetic parameters of homoegonol at the dose of 5 mg/kg. Because the detection of plasma concentrations of homoegonol at 5 mg/kg was shorter than those at 10 and 20 mg/kg, these data were not included in statistical analysis. c Not detected (below detection limit of 1 ng). Cmax, Peak plasma concentration; Tmax, time to reach Cmax; F, bioavailability. b
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Figure 4. Mean plasma concentration–time profiles of homoegonol after its oral administration at 5 (●, n = 4), 10 (▼, n = 7) and 20 (■, n = 5) mg/kg to rats. Bars represent SD.
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(Table 5). The dose-normalized (based on 5 mg/kg) AUCs were also comparable among three doses studied: the values were 6.88 ± 5.54, 8.19 ± 4.77 and 5.67 ± 4.56 ng h/mL for 5, 10 and 20 mg/kg, respectively. Moreover, the slope between the log AUC and log dose (0.8602) was close to 1. Other pharmacokinetic parameters of homoegonol listed in Table 5 were also comparable among three doses studied. The above data indicated that the pharmacokinetic parameters of intravenous homoegonol were also dose-independent. The bioavailability (F) ranged from 0.74 to 1.07% for oral doses studied (Table 5). This low F appears to be due to the extensive metabolism of homoegonol on the basis of the pharmacokinetics and metabolism characteristics of other neolignans such as magnolol and 4O-methylhonokiol. The F of magnolol was shown to be 4% in rats owing to the extensive metabolism via glucuronidation and sulfation (Tsai et al., 1996; Lin et al., 2011). 4-OMethylhonokiol showed high systemic clearance, a short half-life and low oral bioavailability (6.0%) in rats owing to the extensive metabolism via O-demethylation, glucuronidation and sulfation (Yu et al., 2013).
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LC-MS/MS of homoegonol in rat plasma
Conclusion A sensitive, reproducible and selective LC-MS/MS method for the quantification of homoegonol in rat plasma was developed for the first time using protein precipitation as the sample preparation. This study demonstrated the acceptable sensitivity (LLOQ, 1 ng/mL), selectivity, precision, accuracy and stability of the present method. The intravenous and oral pharmacokinetics of homoegonol were evaluated in rats at various doses. The pharmacokinetic parameters of homoegonol were dose-independent after intravenous and oral administration to rats. After oral administration, the bioavailability was very low, 0.74–1.073%.
Acknowledgements This study was supported by the Korea Health 21 R&D Project, the Ministry of Health and Welfare, Republic of Korea (HI12C1852), KRIBB Research Initiative Program (KGM1221413) and The Catholic University of Korea, 2012 (M-2012-B000200024). The authors declare that there is no conflict of interest.
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Copyright © 2014 John Wiley & Sons, Ltd.
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Revised: 15 March 2014,
Accepted: 31 March 2014
Published online in Wiley Online Library: 10 May 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3225
Quantification of homoegonol in rat plasma using liquid chromatography–tandem mass spectrometry and its pharmacokinetics application Hyeon-Uk Jeonga†, Soon Sang Kwona†, Deok-Gyu Hwanga, Eun Nam Kima, Kyeong Leeb, Kyung-Seop Ahnc, Sei-Ryang Ohc and Hye Suk Leea* ABSTRACT: Homoegonol is a biologically active neolignan isolated from Styrax species with cytotoxic, antimicrobial, antiinflammatory and anti-asthma activities. For the quantification of homoegonol in rat plasma, a selective and sensitive liquid chromatography–tandem mass spectrometric method was developed and validated for the first time using protein precipitation with methanol as a sample clean-up procedure. The analytes were separated in an Atlantis dC18 column using a gradient elution of methanol and 0.1% formic acid, and mass-to-charge ratios were determined in selective reaction monitoring mode using tandem mass spectrometry with m/z 343.12 > 296.97 for homoegonol and m/z 517.30 > 282.90 for udenafil (internal standard). The standard curve was linear over the concentration ranges of 1 500 ng/mL using a 30 μL rat plasma sample. The coefficient of variation and relative error for intra- and inter-assay at four quality control levels were 3.9–10.0 and -3.3–2.7%, respectively. The overall recovery of homoegonol from rat plasma using protein precipitation was 99.7 ± 7.7%. The pharmacokinetics parameters of homoegonol were dose-independent after both intravenous (1, 2.5 and 5 mg/kg doses) and oral (5, 10 and 20 mg/kg doses) administration in male Sprague–Dawley rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: homoegonol; liquid chromatography-tandem mass spectrometry; rat plasma; pharmacokinetics
Introduction
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Homoegonol is a biologically active neolignan isolated from Styrax species, such as Styrax camporum, Styrax japonica and Styrax pohlii (Bertanha et al., 2012; Moraes et al., 2012; Teles et al., 2005) and showed cytotoxic (Hirano et al., 1994; Teles et al., 2005), antimicrobial (Ozturk et al., 2008; Pauletti et al., 2000), anti-inflammatory (Bertanha et al., 2012; Moraes et al., 2012; Rodrigues and de Carvalho, 2008) and anti-asthmatic effects in an ovalbumin-induced murine asthma model (Shin et al., 2014). Asthma is a complex chronic inflammatory airway disease with substantial increase in incidence. Numerous drugs have been used for treating asthma, but the use of drugs is limited owing to low efficacy or side effects (Durham et al., 2011). New drug development for the treatment of asthma is urgently needed. Based on our previous results (Shin et al., 2014), homoegonol is currently being evaluated in various efficacy and toxicity studies for development of anti-asthmatic drugs. The evaluation of the bioavailability and pharmacokinetics of homoegonol can link the data from pharmacological assays to clinical effects and design the rational dosage regimens. However, there has been no report on the pharmacokinetics of homoegonol or a bioanalytical method for the quantification of homoegonol in biological fluids. The purpose of the present paper was to develop a rapid, reproducible, selective and sensitive LC-tandem mass spectrometric (LC-MS/MS) method using electrospray ionization for quantification of homoegonol in rat plasma and to characterize
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the pharmacokinetics of homoegonol after intravenous and oral administration at various doses in male Sprague–Dawley rats.
Experimental Materials and reagents Homoegonol was obtained from Toronto Research Chemicals Inc. (Toronto, Canada). Udenafil (used as an internal standard, IS) was a gift from Dong-A Pharmaceutical Co. (Yongin, Korea). Methanol and water (LC-MS grade) were obtained from Fisher Scientific (Pittsburgh, PA, USA). Other chemicals used were of the highest quality available. Drug-free rat
* Correspondence to: Hye Suk Lee, Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea. Email: [email protected] †
The first two authors contributed equally.
a
Drug Metabolism and Bioanalysis Laboratory, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
b
College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea
c
Natural Medicine Research Center, Korea Research Institute of Biology and Biotechnology, Chungbuk 363-883, Republic of Korea Abbreviations used: SRM, selected reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
LC-MS/MS of homoegonol in rat plasma plasma containing sodium heparin as the anticoagulant was prepared from male Sprague–Dawley rats.
Preparation of calibration standards and quality control samples Stock solutions of homoegonol (1 mg/mL) and udenafil (1 mg/mL) were prepared in dimethyl sulfoxide. The stock solution of homoegonol was serially diluted with acetonitrile to working standard solutions. Udenafil stock solution was diluted to 5 ng/mL with methanol. All standard solutions were stored at 4 °C for 4 weeks in amber glass vials in the dark when not in use. Rat plasma calibration standards of homoegonol at concentrations of 1, 2, 5, 10, 50, 200, 400 and 500 ng/mL were prepared by addition of 1.5 μL of the working standard solutions (0.02, 0.04, 0.1, 0.2, 1, 4, 8 and 10 μg/mL) to 30 μL of drug-free rat plasma. To prepare quality control (QC) samples at 1, 3, 30, 375 and 7500 ng/mL, 60 μL of the appropriate working standard solutions at 0.1, 0.3, 3, 37.5 and 750 μg/mL were added to 5940 μL of drug-free rat plasma. Aliquots (30 μL) of QC samples were transferred into polypropylene tubes and stored at 80 °C until analysis.
Sample preparation
Pharmacokinetic study of homoegonol in rats
A 30 μL aliquot of rat blank plasma, calibration standards and QC samples were vortex-mixed with 100 μL of udenafil in methanol (5 ng/mL, IS) for 3 min at high speed. After centrifugation at 13,000 rpm at 4 °C for 8 min, 50 μL of the supernatant was diluted with 50 μL of water. The aliquot (7 μL) was injected into the LC-MS/MS.
LC-MS/MS analysis The LC-MS/MS system consisted of a Nanospace SI-2 nano-LC (Shiseido, Tokyo, Japan) coupled with a Finnigan Quantum Access tandem mass spectrometer (Thermo Scientific, San Jose, CA, USA). Separation was performed on the Atlantis dC18 column (5 μm, 2.1 mm i.d. × 100 mm, Waters Co.) using gradient elution of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.3 mL/min: 40% mobile phase B for 0.5 min, 40% to 95% mobile phase B for 0.1 min, 95% mobile phase B for 4.9 min, 95–40% mobile phase B for 0.1 min, 40% mobile phase B for 4.4 min. The column and autosampler were maintained at 50 and 6 °C, respectively. The electrospray ionization source settings for analysis of homoegonol and udenafil were as follows: spray voltage, 4.5 kV; vaporizer temperature, 350 °C; capillary temperature, 330 °C; sheath gas pressure, 35 psi; and auxiliary gas pressure, 15 psi. The tube lens offsets for homoegonol and udenafil were 60 and 94 V, respectively. Fragmentation + of the [M + H] ion for homoegonol and udenafil was performed at a collision energy of 19 and 40 V, respectively, by collision-activated dissociation with argon gas as the collision gas at a pressure setting of 1.3 on the instrument. Selected reaction monitoring (SRM) mode was employed for the quantification: m/z 343.12 → 296.97 for homoegonol and m/z 517.30 → 282.90 for udenafil. Xcalibur® software (ThermoFisher Scientific) was used for the LC-MS/MS system control and data processing.
Method validation
This method was applied to the pharmacokinetic study of homoegonol after intravenous and oral administration at various doses in male Sprague–Dawley rats (body weight 229 263 g) (Samtako Co., Osan, Korea). The animal experiments were approved by institutional Animal Ethical Committee. Animals were kept in plastic cages with free access to standard rat diet (Samtako Co.) and water before the experiment. Animals were maintained at a temperature of 22 24 °C with a 12 h light/dark cycle and relative humidity of 50 ± 10%. Rats were anesthetized by isoflurane and were cannulated with polyethylene tubing (PE-50, Natsume Co., Tokyo, Japan) in the jugular vein for blood sampling and in the femoral vein for intravenous injection. Each rat was housed individually in a rat metabolic cage and allowed to recover from anesthesia for 1 day prior to the start of the study. Rats were not restrained at any time during the study. In order to prevent blood clotting, each catheter was flushed with heparin in physiological saline solution (10 U/mL). Homoegonol was dissolved in a mixture of N,N-dimethylacetamide, Tween 80 and saline (6:1:8, v/v) and was administered to rats by bolus injection via the femoral vein at 1 (n = 3), 2.5 (n = 4) and 5 (n = 4) mg/kg doses. For the oral study, homoegonol (the same solution used in the intravenous study) was administered to rats by stomach gavage tube at doses of 5 (n = 4), 10 (n = 7) and 20 mg/kg (n = 5). Blood samples (150 μL) were collected before (control) and at the following time points following drug administration: 2 (only for intravenous), 5, 15, 30, 45, 60 min, and at 1.5, 2, 3, 4, 6, 8, 12 and 24 h. Plasma samples were harvested by centrifugation at 13,000 rpm for 8 min and stored at 80 °C until analysis. The pharmacokinetic parameters of homoegonol were analyzed by a noncompartment analysis (WinNonlin, Pharsight, Mountain View, CA, USA): the total area under the plasma concentration–time curve (AUC), the terminal half-life (t1/2), the time-averaged total body, renal and nonrenal clearances (Cl, Clr, and Clnr, respectively), and the apparent volume of distribution at steady-state (Vss). The peak plasma concentration (Cmax) and the time to reach Cmax (Tmax) were directly obtained from the experimental data. The bioavailability (F) values were calculated by dividing dose-normalized (at 2.5 mg/kg) AUC obtained after oral administration by the AUC after the intravenous administration at a dose of 2.5 mg/kg. All data are expressed as the means ± SD.
Results and discussion LC-MS/MS Electrospray ionization of homoegonol and udenafil produced abundant [M + H]+ ions at m/z 343.12 and 517.30, respectively,
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In order to complete the method validation, the batches consisting of triplicate calibration standards covering the range of 1-500 ng/mL and five replicates of QC samples at 1, 3, 30, and 375 ng/mL were analyzed on three consecutive days. The relative error (RE), the percentage of deviation of the mean from the true values, served as a measure of the accuracy, and the coefficient of variation (CV) indicated the precision. To demonstrate selectivity, rat plasma from 10 different rats was screened for interference at the retention time and mass transition of homoegonol and udenafil (IS). The matrix effect for homoegonol and udenafil (IS) was investigated according to the bioanalysis method validation guideline of the
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European Medicines Agency. The matrix factor was evaluated by calculating the ratio of the peak area of analyte spiked after extraction into plasma extracts originating from six different rats to the peak areas for the pure solution of the analyte in 40% methanol at 1, 3 and 375 ng/mL. The IS-normalized matrix factor was calculated by dividing the matrix factor of homoegonol by the matrix factor of the IS. The CV of the IS-normalized matrix factor from the six different rat plasmas should not be greater than 15%. Recovery of homoegonol and udenafil was determined by comparing the mean peak areas of the analytes spiked before extraction into the blank plasma sources with those of the analytes spiked post-extraction into the blank plasma extracts at four concentrations, which were 1, 3, 30 and 375 ng/mL. QC samples at 7500 ng/mL were diluted 20-fold with blank rat plasma to establish the concentration above the upper limit of the calibration curve that can be diluted into quantifiable range. For evaluation of freeze–thaw stability, long-term storage stability, short-term storage stability and room temperature storage stability, five replicates of the QC samples at low and high concentrations (3 and 375 ng/mL) were subjected to three freeze–thaw cycles, storage at 80 °C for 25 days, or storage at room temperature for 2 h before processing. Post-extraction batch integrity was determined by batch reinjection after 24 h of storage in the autosampler.
H.-U. Jeong et al. without evidence of fragmentation or adduct formation. The [M + H]+ ion of homoegonol and udenafil was selected as the precursor ion and was subsequently fragmented in MS/MS mode in order to obtain the product ion spectra, yielding useful structural information (Fig. 1). Homoegonol produced the major product ion at m/z 296.97 by loss of ethanol from the [M + H]+ ion, and udenafil showed a prominent product ion at m/z 282.90 [5-(2-hydroxy-phenyl)-1-methyl-3-propyl-1,4-dihydropyrazolo[4,3-d]pyrimidin-7-one radical]. For the quantification of homoegonol in the plasma, SRM mode was used owing to the high selectivity and sensitivity of SRM data acquisitions, where the transition of the precursor ion to a product ion was monitored as follows: m/z 343.12 to m/z 296.97 for homoegonol and m/z 517.30 to m/z 282.90 for udenafil. Homoegonol showed better retention and peak shape on an Atlantis dC18 column compared with a Pinnacle biphenyl column and octadecyl-bonded phase columns such as Imtakt C18 and Halo C18. Use of methanol as an organic solvent in the mobile phase increased the organic solvent content compared with acetonitrile and resulted in high sensitivity of homoegonol owing to the increase of electrospray ionization efficiency.
Method validation Calibration curves were obtained over the concentration ranges of 1–500 ng/mL of homoegonol in rat plasma. Linear regression analysis with a weighting of 1/concentration gave the optimum accuracy (RE, 2.0–5.1%) and precision (CV, ≤13.2%) of the corresponding calculated concentrations at each level (Table 1). The low CV value (7.4%) for the slope indicated the repeatability of the method (Table 1). Table 2 shows a summary of the intra- and inter-day precision and accuracy data for QC samples containing homoegonol. Both intra- and inter-day CV values ranged from 3.9 to 10.0% at four QC levels. Intra- and inter-assay RE values were 3.3 to 2.7% at four QC levels. These results indicated acceptable accuracy and precision of the present method. The lower limit of quantification (LLOQ) for homoegonol was set at 1 ng/mL using 30 μL of rat plasma with a signal-to-noise ratio >10 (Fig. 2b). After a 20-fold dilution of the dilution quality control samples at 7500 ng/mL, the CV and RE for homoegonol were 5.0 and 5.8%, respectively, indicating the acceptability of the 20-fold dilution prior to analysis. In the analysis of the blank plasma samples obtained from 10 different rats, no interference peak was observed at the retention times of homoegonol (3.63 min) and udenafil (3.03 min), indicating the selectivity of the present method (Fig. 2a). Sample carryover effect was not observed. The matrix factor for homoegonol at 1, 3 and 375 ng/mL was 0.96 ± 0.09, 0.94 ± 0.06 and 0.96 ± 0.03, respectively. The matrix factor for IS was 0.91 ± 0.06. The IS-normalized matrix factor for homoegonol at 1, 3, and 375 ng/mL was 1.08 ± 0.06, 1.09 ± 0.06, and 1.01 ± 0.05, respectively. The CV values of the IS-normalized matrix factor from the six different rat plasmas were 5.9, 5.2 and 4.9% for 1, 3 and 375 ng/mL, respectively. These data confirm that the matrix effects for homoegonol and udenafil have little effect on the determination of homoegonol.
Table 2. Precision and accuracy of homoegonol in quality control (QC) samples Statistical variable
Figure 1. Product ion mass spectra of (a) homoegonol and (b) udenafil (internal standard).
Intra-day (n = 5)
Inter-day (n = 3)
QC (ng/mL) 1.0 3.0 30.0 375.0 1.0 3.0 30.0 375.0 Mean 1.0 2.9 30.8 368.5 1.0 2.9 30.3 376.8 (ng/mL) CV (%) 10.0 4.7 5.2 5.2 9.4 7.8 4.3 3.9 RE (%) 0.0 –3.3 2.7 –1.7 0.0 –3.3 1.0 0.5
Table 1. Calculated concentrations of homoegonol in calibration standards prepared with rat plasma (n = 9) Statistical variable Mean (ng/mL) CV (%) RE (%)
Theoretical concentration (ng/mL)
Slope
1.0
2.0
5.0
10.0
50.0
200.0
400.0
500.0
1.0 13.2 0.0
2.0 2.0 0.0
4.9 9.6 –2.0
9.8 4.1 –2.0
50.9 1.5 1.8
210.2 1.0 5.1
392.0 3.1 –2.0
497.3 2.7 –0.5
0.0889 7.4
Intercept
0.0094
r2
0.9983 0.1
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CV, Coefficient of variation; RE, relative error
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LC-MS/MS of homoegonol in rat plasma
Figure 2. Selected reaction monitoring chromatograms of (a) a rat blank plasma, (b) a rat plasma sample spiked with 1 ng/mL of homoegonol, and (c) a rat plasma sample obtained 15 min after intravenous administration of homoegonol at a dose of 2.5 mg/kg to a male Sprague–Dawley rat.
The overall recovery of homoegonol from rat plasma using protein precipitation was 99.7 ± 7.7% for four QC concentration levels and was consistent over the concentration range of 1–375 ng/mL. Recovery of the internal standard udenafil was 91.4 ± 3.8%. The stability of processing (freeze–thaw, long-term storage at 80 °C, and short-term storage at room temperature) and chromatography (re-injection) was assessed and was shown to be of insignificant effect (Table 3). Three freeze–thaw cycles, long-term storage at 80 °C for 25 days, and room temperature storage of QC samples for 2 h at low and high concentrations prior to analysis had little effect on quantification. Re-analysis of the organic extracts stored for 24 h at 6 °C showed acceptable accuracy and precision of the QC samples. Pharmacokinetics of homoegonol in rats The method was applied successfully to study pharmacokinetics after intravenous injection of homoegonol at 1, 2.5 and 5 mg/kg doses and oral administration of homoegonol at 5, 10 and
Table 3. Stability of quality control samples (n = 5) Statistical variable
20 mg/kg doses in male Sprague–Dawley rats. Figure 2(c) shows representative SRM chromatograms from the analysis of a plasma sample obtained 15 min after intravenous administration of homoegonol at 2.5 mg/kg dose in a rat. After intravenous administration of homoegonol, its mean plasma concentration–time curves declined in a poly-exponential fashion for all three doses studied (Fig. 3). Its AUCs were linearly increased as the dose increased (Table 4). The dose-normalized (based on 1 mg/kg) AUCs were comparable among three doses studied: the values were 126.4 ± 9.8, 152.6 ± 32.5 and 132.6 ± 38.2 ng h/mL for 1, 2.5, and 5 mg/kg, respectively. Moreover, the slope between the log AUC and log dose (1.0388) was close to 1. The other pharmacokinetic parameters of homoegonol shown in Table 4 were also comparable among three doses studied, indicating that the pharmacokinetic parameters of intravenous homoegonol were independent of dose. The contribution of the renal clearance to the clearance of homoegonol was negligible: Ae0–24h was 0.001–0.006% for all three doses studied. This indicated that homoegonol is almost completely eliminated via the nonrenal route. After oral administration, homoegonol was rapidly absorbed and was detected at the first blood sampling time point (5 min) with rapid Tmax, 0.22–0.50 h for all three doses (Fig. 4, Table 5). As the oral dose increased, AUC and Cmax were linearly increased
Theoretical concentration (ng/mL) 3.0
375.0
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Figure 3. Mean plasma concentration–time profiles of homoegonol after its intravenous administration at 1 (●, n = 3), 2.5 (▼, n = 4) and 5 (■, n = 4) mg/kg to rats. Bars represent SD.
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Three freeze–thaw cycles Mean (ng/mL) 2.8 371.7 CV (%) 3.4 9.4 RE (%) –6.7 –0.9 Short-term stability (2 h storage at room temperature) Mean (ng/mL) 3.2 365.3 CV (%) 4.7 2.4 RE (%) 6.7 –2.6 Long-term storage stability (25 days at –80 °C) Mean (ng/mL) 2.7 343.6 CV (%) 4.9 1.6 RE (%) –10.0 –8.4 Post-preparative stability (24 h in autosampler) Mean (ng/mL) 3.0 390.9 CV (%) 7.6 2.1 RE (%) 0.0 4.2
H.-U. Jeong et al. Table 4. Mean pharmacokinetic parameters of homoegonol after its intravenous administration at various doses to male rats Parameter
1 mg/kg (n = 3) a
AUC (ng h/mL) Cl (mL/min/kg) Clr (mL/min/kg) Clnr (mL/min/kg) Vss (L/kg) t1/2 (h) Ae0–24h (percentage of the dose)
126.4 ± 9.8 130.25 ± 9.63 0.006 ± 0.005 130.25 ± 9.63 3.11 ± 0.43 (0.49 ± 0.13)b 0.005 ± 0.004
2.5 mg/kg (n = 4)
5 mg/kg (n = 4)
381.4 ± 81.3 112.69 ± 25.35 0.007 ± 0.002 112.69 ± 25.35 2.92 ± 0.37 0.88 ± 0.20 0.006 ± 0.001
662.8 ± 191.1 133.02 ± 36.92 0.002 ± 0.003 133.02 ± 36.92 4.84 ± 1.52 1.29 ± 0.38 0.001 ± 0.002
a
Dose-normalized (1 mg/kg) AUCs were compared for statistical analysis. The numbers in parentheses represent pharmacokinetic parameters of homoegonol at the dose of 1 mg/kg. Because the detection of plasma concentrations of homoegonol at 1 mg/kg was shorter than those at 2.5 and 5 mg/kg, these data were not included in statistical analysis. AUC, total area under the plasma concentration-time curve; t1/2, the terminal half-life; Cl, Clr, and Clnr, the time-averaged total body, renal and nonrenal clearances, respectively; Vss, the apparent volume of distribution at steady-state; Ae0–24h, the percentage of the dose of homoegonol excreted in 24 h urine. b
Table 5. Mean pharmacokinetic parameters of homoegonol after its oral administration at various doses to rats Parameter AUC (ng h/mL)a Tmax (h) Cmax (ng/mL)a t1/2 (h) Ae0–24h (percentage of the dose) F (%)
5 mg/kg (n = 4)
10 mg/kg (n = 7)
20 mg/kg (n = 5)
6.88 ± 5.54 0.50 ± 0.20 9.1 ± 5.3 (0.50 ± 0.26)b NDc 0.90 ± 0.73
16.37 ± 9.54 0.50 ± 0.20 19.2 ± 6.0 0.49 ± 0.16 0.003 ± 0.003 1.07 ± 0.62
22.67 ± 18.23 0.22 ± 0.07 27.1 ± 17.3 0.69 ± 0.28 0.004 ± 0.008 0.74 ± 0.60
a
Dose-normalized (5 mg/kg) AUCs and Cmax were compared for statistical analysis. The numbers in parentheses represent pharmacokinetic parameters of homoegonol at the dose of 5 mg/kg. Because the detection of plasma concentrations of homoegonol at 5 mg/kg was shorter than those at 10 and 20 mg/kg, these data were not included in statistical analysis. c Not detected (below detection limit of 1 ng). Cmax, Peak plasma concentration; Tmax, time to reach Cmax; F, bioavailability. b
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Figure 4. Mean plasma concentration–time profiles of homoegonol after its oral administration at 5 (●, n = 4), 10 (▼, n = 7) and 20 (■, n = 5) mg/kg to rats. Bars represent SD.
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(Table 5). The dose-normalized (based on 5 mg/kg) AUCs were also comparable among three doses studied: the values were 6.88 ± 5.54, 8.19 ± 4.77 and 5.67 ± 4.56 ng h/mL for 5, 10 and 20 mg/kg, respectively. Moreover, the slope between the log AUC and log dose (0.8602) was close to 1. Other pharmacokinetic parameters of homoegonol listed in Table 5 were also comparable among three doses studied. The above data indicated that the pharmacokinetic parameters of intravenous homoegonol were also dose-independent. The bioavailability (F) ranged from 0.74 to 1.07% for oral doses studied (Table 5). This low F appears to be due to the extensive metabolism of homoegonol on the basis of the pharmacokinetics and metabolism characteristics of other neolignans such as magnolol and 4O-methylhonokiol. The F of magnolol was shown to be 4% in rats owing to the extensive metabolism via glucuronidation and sulfation (Tsai et al., 1996; Lin et al., 2011). 4-OMethylhonokiol showed high systemic clearance, a short half-life and low oral bioavailability (6.0%) in rats owing to the extensive metabolism via O-demethylation, glucuronidation and sulfation (Yu et al., 2013).
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LC-MS/MS of homoegonol in rat plasma
Conclusion A sensitive, reproducible and selective LC-MS/MS method for the quantification of homoegonol in rat plasma was developed for the first time using protein precipitation as the sample preparation. This study demonstrated the acceptable sensitivity (LLOQ, 1 ng/mL), selectivity, precision, accuracy and stability of the present method. The intravenous and oral pharmacokinetics of homoegonol were evaluated in rats at various doses. The pharmacokinetic parameters of homoegonol were dose-independent after intravenous and oral administration to rats. After oral administration, the bioavailability was very low, 0.74–1.073%.
Acknowledgements This study was supported by the Korea Health 21 R&D Project, the Ministry of Health and Welfare, Republic of Korea (HI12C1852), KRIBB Research Initiative Program (KGM1221413) and The Catholic University of Korea, 2012 (M-2012-B000200024). The authors declare that there is no conflict of interest.
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