Journal of Chromatography B, 969 (2014) 249–255
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Plasma pharmacokinetics and tissue distribution study of roemerine in rats by liquid chromatography with tandem mass spectrometry (LC–MS/MS) Yue-Qiong Liu a,b , Gong-Hao He a , Hong-Liang Li c , Jiang-Chang He a , En-Fu Feng a , Lan Bai a , Cheng-Ying Wang a , Gui-Li Xu a,∗ a
Department of Pharmacy, Kunming General Hospital of Chengdu Military Region, Kunming 650032, PR China Department of Pharmacy, Wenshan city People’ Hospital, Wenshan 663000, PR China c Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China b
a r t i c l e
i n f o
Article history: Received 10 June 2014 Received in revised form 22 August 2014 Accepted 25 August 2014 Available online 28 August 2014 Keywords: Roemerine Pharmacokinetics Tissue distribution LC–MS/MS
a b s t r a c t In the present study, a new LC–MS/MS method for the determination of roemerine in rat plasma and tissue samples was developed and successfully used to study the pharmacokinetics and tissue distribution of roemerine after oral and intravenous (i.v.) administration in rats. The plasma and tissue samples were processed by liquid–liquid extraction with n-hexane. Isocorydine was used as the internal standard (IS) for sample processing and analysis. The MS/MS detection was carried out by monitoring the transitions of m/z 280→249 and m/z 342→279 for roemerine and the IS, respectively. The calibration curve displayed excellent linearity over the concentration range of 10–2000 ng/mL (n = 8, r2 ≥ 0.995), and the lower limit of quantification (LLOQ) was determined to be 10 ng/mL. This method was rapid, accurate, highly sensitive, and fully validated. The pharmacokinetic study showed that roemerine was rapidly absorbed and eliminated with a tmax of 0.22 ± 0.08 h, t1/2 of 1.59 ± 0.46 h, CL of 4.44 ± 0.42 L/h/kg, and Vd of 10.16 ± 2.95 g/L following oral administration. Additionally, roemerine showed an excellent oral bioavailability of 84% and a wide tissue distribution with brain penetration. Highest concentrations of roemerine were found in the liver and lung, followed by kidney, spleen, heart, and brain, in that order. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Roemerine (Fig. 1A) is an aporphine alkaloid found in many plants such as Nelumbo nucifera [1,2], Stephania kwangsiensis [3], Turkish Papaver [4], Rollinia leptopetala [5], Hornschuchia oblique [6], and Neolitsea dealbata [7]. Roemerine has also been produced by biosynthesis [8]. Pharmacological studies have shown that roemerine possesses diverse beneficial bioactivities. It is a vasodilator that inhibits the noradrenaline-induced contraction [9,10]. It also interacts with the P-glycoprotein and enhances the cytotoxicity of vinblastine against multidrug-resistant KB-V1 cells [11]. In addition, it binds to the 5-HT2A receptor with high affinity, which suggests that it may have CNS effects [12,13]. Roemerine also exhibits antiplasmodial [14], antibacterial [15], antifungal [16], and anthelmintic [17] activities. Moreover, roemerine regulates glucose metabolism by stimulating glucose consumption and inhibiting glucose absorption [18,19]. Therefore, roemerine may have potential value as
therapeutic agent for a variety of diseases. Nonetheless, in contrast to the comprehensive research on bioactivity, to date the pharmacokinetic characteristics of roemerine have not been reported. Roemerine in raw plant extracts has been determined using high performance liquid chromatography (HPLC) coupled with a diode array detector [20,21]. However, this method is not suitable for pharmacokinetic studies of roemerine, which require pretreatment of samples and higher sensitivity levels of detection. LC–MS/MS is a well-established analytical method with high specificity, high sensitivity, and high throughput [22]. In the present work, we first developed and validated a new LC–MS/MS method for determination of roemerine in rat plasma and tissue samples. This method was successfully used to study the pharmacokinetics, oral bioavailability, and tissue distribution of roemerine in rats. 2. Experimental 2.1. Chemicals and reagents
∗ Corresponding author. Tel./fax: +86 871 4774784. E-mail address: [email protected] (G.-L. Xu). http://dx.doi.org/10.1016/j.jchromb.2014.08.031 1570-0232/© 2014 Elsevier B.V. All rights reserved.
Roemerine (purity 97%) and isocorydine (purity 98%; Fig. 1B) were kindly provided by Professor Gao-Xiong Rao of the School
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Fig. 1. Chemical structures and product ion spectra of roemerine (A) and isocorydine (B) in positive mode.
of Pharmacy, Yunnan College of Traditional Chinese Medicine. Methanol, acetic acid, and n-hexane (all HPLC grade) were purchased from Merck KGaAS (Darmstadt, Germany). Distilled, deionized water freshly generated on a Milli-Q reagent water system (Millipore, USA) was used in all sample preparations and chromatography. All other reagents were of analytical grade and purchased from commercial sources. 2.2. LC–MS/MS instrument and conditions The HPLC was performed on an Agilent 1200 series HPLC system (Agilent, USA) equipped with an autosampler, a degasser, and two SL bin-pumps. The chromatographic separation was achieved on a symmetry C18 analytical column (50 mm × 2.1 mm, 5 m) connected to a SecurityGuard Cartridge AQC18 guard column (30 mm × 4 mm, 5 m), thermostated at 35 ◦ C. The samples (10 L each) were run using an isocratic mobile phase of methanol–water mixture (50:50, v/v) containing 0.1% acetic acid, at a flow rate of 0.2 mL/min. The Agilent 1200 HPLC system was coupled on-line to a G6410B triple quadruple mass spectrometer (Agilent, USA) equipped with an electrospray ionization (ESI) source. The mass spectrometer was operated using the ESI positive ion mode under multiplereaction monitoring (MRM). The optimized precursor-to-product ion transitions were monitored at m/z 280→249 and m/z 342→279 for roemerine and the IS, respectively. The optimized parameters for MS analysis were: capillary voltage 4000 V; nebulizer pressure 35 psi; dry gas temperature 350 ◦ C; gas flow 10 L/min; collision energies (CE) 13 eV; fragmentor potential 78 V and 86 V for roemerine and the IS, respectively. 2.3. Standard and quality control (QC) sample preparation The stock solutions of roemerine and IS were both prepared in methanol at a final concentration of 1 mg/mL. Working solutions
of roemerine (100–20000 ng/mL) and the IS (2000 ng/mL) were obtained by diluting the stock solutions with methanol–H2 O (50:50, v/v). All of the solutions were kept at −4 ◦ C until analysis. The calibration standards of roemerine (10–2000 ng/mL) were prepared by adding 10 L of the working solution at different concentrations to 90 L of the blank biological matrix. QC samples of roemerine (20, 200, and 1600 ng/mL) were similarly prepared. 2.4. Sample preparation In the present study, roemerine and the IS were extracted from rat plasma and tissue homogenates using a simple liquid–liquid extraction (LLE) method. After samples were thawed at room temperature, an aliquot of 100 L plasma or tissue homogenates was transferred into a 2 mL tube. The sample was first vortex-mixed with 10 L of IS working solution and then with 1 mL of n-hexane for extraction. After vortex for 2 min and centrifuged at 10,000 rpm for 5 min, the supernatant was transferred into another tube and evaporated to dryness under a gentle stream of nitrogen at 25 ◦ C. The residue was reconstituted with 100 L methanol–H2 O (50:50, v/v) and subjected to LC–MS/MS analysis. 2.5. Method validation The developed method was validated according to the FDA guidelines for the validation of bioanalytical methods [23]. 2.5.1. Specificity Specificity of the method was assessed by analyzing blank biological samples from six different sources, blank biological samples spiked with roemerine and the IS, and plasma and tissue samples collected after oral administration of roemerine, which were also spiked with the IS.
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2.5.2. Linearity and LLOQ The calibration curves were generated by plotting the peak area ratios of roemerine to the IS (y-axis) versus nominal concentrations of roemerine (x-axis) using a weighted linear regression (1/x2 ). The LLOQ was defined as the lowest concentration of roemerine in the calibration curve that could be detected with an acceptable precision not exceeding 20% and an accuracy within ±20%. 2.5.3. Accuracy and precision The intraday accuracy and precision were determined by analyzing six replicates of the low, medium, and high QC samples (20, 200, and 1600 ng/mL, respectively). The interday accuracy and precision were determined over test results in three consecutive days. An accuracy within ±15% of the relative error (RE) from the nominal values and a precision ≤15% of the relative standard deviation (RSD) were deemed to be acceptable. 2.5.4. Recovery and matrix effect The extraction recovery in rat sample matrices for roemerine and the IS was calculated as the peak area ratios of the rat sample matrix spiked with a standard solution to the blank matrix spiked with an equivalent standard solution. The recovery of roemerine was determined at low, medium and high concentrations, while the recovery of the IS was determined at a single concentration of 200 ng/mL. The matrix effect of extraction on roemerine analysis was evaluated by comparing the peak areas of the n-hexane extracted blank samples spiked with roemerine at three QC concentrations with those of roemerine standard solutions at equivalent concentrations. The matrix effect of extraction on IS analysis was similarly evaluated. 2.5.5. Stability The stability of roemerine in rat biological samples was studied using blank biological samples spiked with QC solutions at low, medium, and high concentrations in triplicate. The stability of samples was analyzed under four different conditions: (1) after 20 days of storage at −20 ◦ C (long-term stability); (2) after 4 h at room temperature (short-term stability); (3) after three freeze–thaw (−20 ◦ C/room temperature) cycles; and (4) after 24 h in the autosampler (post-preparation stability). The stability was deemed acceptable when the percentage deviation was within ±15%. 2.5.6. Dilution integrity The blank liver and lung samples spiked with 6400 ng/mL of roemerine (exceeding the upper limit of the calibration curve) were diluted by four-fold and subjected to LC–MS/MS analysis. The experiments were conducted in six replicates. The dilution integrity was deemed acceptable when the precision and accuracy were within ±15%. 2.5.7. Carryover evaluation Carryover of roemerine was assessed by injection of blank samples (no roemerine and the IS) directly after injection of the highest point in the calibration curve (2000 ng/mL). Carry over in the blank sample following the high concentration standard should not be greater than 20% of the LLOQ and 5% for the internal standard. 2.6. Animals The animal study protocol was approved by the Institutional Animal Care and Use Committee of Kunming General Hospital of Chengdu Military Region. Sprague–Dawley rats (200 ± 20 g, both male and female) were purchased from the Central Animal House of Kunming Medical University and acclimated in the laboratory for
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1 week prior to the experiments. All of the rats were housed and cared for under constant temperature of 23 ± 1 ◦ C and humidity of 60 ± 10% with free access to food and water. Rats were fasted for 12 h with free access to water prior to roemerine administration. 2.7. Pharmacokinetic study For pharmacokinetic studies, the rats were randomly divided into two groups (n = 6 for each group). Roemerine [injectable solution in ethanol/Tween 80/0.9% NaCl (1:0.2:8.8, v/v/v)] was administered at an oral dose of 20 mg/kg or an intravenous dose of 6 mg/kg. At specific time points after administration (oral: 5 min to 24 h; i.v.: 0 min to 12 h), blood samples (500 L) were collected from the epicanthic veins of rats into heparinized tubes. Samples were immediately centrifuged at 10,000 rpm for 5 min and the supernatant plasma samples were collected and stored at −20 ◦ C until analysis. 2.8. Tissue distribution study The rats were randomly divided into five groups (n = 6 for each group). The rats were sacrificed at 5 min, 20 min, 45 min, 2 h, and 8 h, respectively, after oral administration of roemerine (20 mg/kg). The liver, heart, spleen, lung, kidney, and brain tissues were collected immediately, rinsed with physiological saline solution, blotted dry with filter paper, and homogenized in physiological saline solution (1:2, w/v) with Superfine Homogenizer. The tissue homogenates were centrifuged and aliquots of the supernatants were collected and stored at −20 ◦ C until analysis. 2.9. Data analysis The plasma and tissue homogenates were subjected to LC–MS/MS analysis as above and the concentrations of roemerine were calculated according to the calibration curves. The pharmacokinetic parameters including the area under the curve (AUC), half-life (t1/2 ), apparent central volume of distribution (V), and clearance rate (CL) were calculated by the DAS 3.0 software (Drug and Statistics, Mathematical Pharmacology Professional Committee of China, Shanghai, China) with a noncompartmental model. The maximum plasma concentration (Cmax ) and the time to reach Cmax (tmax ) were calculated from the experimental data. Absolute bioavailability (F) was calculated based on the AUC0-∞ of i.v. and oral administration at equivalent doses. All data are presented as means ± standard deviation (SD). 3. Results and discussion 3.1. LC–MS/MS conditions In order to identify the optimal conditions for the determination of roemerine and the IS, we investigated a variety of mobile phase compositions and elution flow rates. We tested mobile phases with varying organic solvent/water compositions and different pH modifiers, the test results showed that the optimal retention time and ionization of roemerine and IS were obtained using the mobile phase of methanol–water (50:50, v/v) containing 0.1% acetic acid. Moreover, we tested elusion flow rate in the range of 0.15–0.4 mL/min, and the flow rate at 0.2 mL/min was selected for sample analysis based on performance. Under the optimized conditions, we did not detect any significant endogenous interference. We tested the positive- and negative-ion ESI modes for determination of roemerine in biological samples. In the positive mode, roemerine showed a strong response of protonated molecular ions [M + H]+ at m/z 280, the signal to noise ratio (s/n) is far higher than
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Fig. 2. Representative chromatograms of blank plasma (A) and blank liver homogenates (B), blank plasma spiked with roemerine (10 ng/mL) and IS (200 ng/mL) (C) and blank liver homogenates spiked with roemerine (10 ng/mL) and IS (200 ng/mL) (D), real plasma sample (491.14 ng/mL) (E) and real liver homogenates (742.93 ng/mL) (F) after oral administration of 20 mg/kg roemerine spiked with IS.
that in negative ions. In addition, the fragmentation in ESI+ is usually richer than that in ESI− . Therefore, the ESI positive ion mode was used for subsequent sample analysis. The collision induced dissociation (CID) parameters were optimized under the MRM mode for stable ion response and high detection sensitivity. Further, the most suitable conditions for mass spectrometry were determined by optimizing collision energy, fragmentor potential, source temperature, drying gas temperature, drying gas flow, nebulizer pressure, and capillary voltage. Under the optimized conditions, the peaks with the highest intensity in the MS spectra of roemerine and the IS were the ions at m/z 280 [M + H]+ and m/z 342 [M + H]+ , respectively. After fragmentation in the collision cell, the most abundant and stable product ions were at m/z 249 and m/z 279 (Fig. 1), respectively.
20% for roemerine, whilst the SPE method suffered a low recovery (approximately 65%). The LLE method demonstrated the best performance among the three methods, and was thus selected as the method for sample pretreatment. We subsequently tested several organic solvents including ethyl acetate, dichloromethane, methyl t-butyl ether, and n-hexane as the extraction solvent in the LLE method for plasma and tissue sample pretreatment. We found that the best overall result (high recovery, low matrix suppression, and fast solvent evaporation) was obtained when n-hexane was used as the extraction solvent. The LLE sample pretreatment with nhexane was quick, convenient, and effective, and only required small amounts of solvent and biological matrix.
3.2. Selection of internal standard
3.4.1. Specificity Fig. 2 shows representative chromatograms of blank matrices, blank matrices spiked with roemerine and the IS, and plasma and tissue samples collected after oral administration of roemerine, which were also spiked with the IS. The retention times of roemerine and the IS were approximately 0.9 min and 0.6 min, respectively. The peaks were well separated with no detectable interfering peaks nearby when blank plasma or tissue homogenate samples spiked with roemerine and the IS were analyzed.
For a proper internal standard, a similar chemical behavior and a suitable retention time were of significant importance. In addition, an analog has to be used because labeled roemerine was not commercially available. Isocorydine, an analog to roemerine, was chosen as the IS because it has similar chemical structure, chromatographic behavior, and MS characteristics to roemerine.
3.4. Method validation
3.3. Optimization of sample pretreatment We tested sample preparation by LLE, solid-phase extraction (SPE), and protein precipitation (PPT). The PPT method showed severe matrix suppression, with a matrix effect of approximately
3.4.2. Linearity and LLOQ Calibration curves were established over the concentration range between 10 and 2000 ng/mL for roemerine in all biological matrices. The calibration curves in all biological matrices
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Table 1 Precision and accuracy of roemerine in plasma and tissue homogenates of rats (n = 6). Biosamples
QC conc (ng/mL) 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
Precision (RSD, %) Intra-day
Inter-day
Accuracy (RE, %) Intra-day
Inter-day
4.36 3.05 1.08 1.82 5.12 3.50 2.52 4.56 3.51 5.12 4.20 3.36 2.89 5.58 4.61 3.97 3.21 4.54 4.33 1.48 3.03
4.23 5.31 2.13 5.82 3.46 5.60 3.65 2.74 3.89 2.65 2.71 2.06 4.02 4.86 3.46 3.43 6.46 1.74 1.03 4.59 0.99
–0.85 0.61 –6.50 6.95 0.50 –5.97 4.87 1.29 –5.64 4.61 –1.07 –5.49 2.60 –0.65 –7.63 2.41 –7.96 –5.46 3.00 6.10 –5.70
2.29 0.51 –5.60 1.28 –1.68 –6.41 2.84 1.87 –5.91 2.13 0.61 –3.24 2.15 –0.25 –6.90 5.90 –0.62 –4.04 1.89 1.25 –5.35
exhibited excellent linearity (r2 ≥ 0.995). The LLOQ of roemerine was determined to be 10 ng/mL (S/N ≥ 10) in all biological matrices, the precision and accuracy of LLOQ were less than 10.55% and within ± 9.09%. 3.4.3. Accuracy and precision The intra- and inter-day accuracy and precision for roemerine in biological samples are summarized in Table 1. The relative errors (RE, %) of assay accuracy ranged from −7.96 to 6.95%, and the precision values (RSD, %) were within 6.64%. These results were all within the threshold of ±15%, which indicated that the method had acceptable accuracy, precision, and reproducibility. 3.4.4. Recovery and matrix effect The extraction recovery and matrix effect for roemerine in plasma and tissue matrices are shown in Table 2. These data indicated reproducible and acceptable recoveries in all biological matrices with no significant ion suppression or enhancement effects. Table 2 Matrix effect and recovery of roemerine in plasma and tissue homogenates of rats (n = 6). Biosamples
Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
QC conc (ng/mL)
20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
Recovery (%)
Matrix effect (%)
Mean
RSD
Mean
RSD
85.22 89.42 87.57 82.60 83.55 85.55 82.55 84.88 80.13 82.18 84.15 82.98 86.67 84.37 84.03 70.88 69.05 69.33 78.33 76.52 75.82
2.56 2.53 3.58 4.42 3.32 4.74 4.38 4.94 3.76 4.84 3.55 2.87 2.32 2.69 3.49 2.62 5.23 5.49 4.70 3.79 3.43
90.53 88.06 86.17 100.27 105.45 107.45 99.35 90.62 92.65 100.05 98.32 104.17 100.57 87.95 93.28 85.1 91.48 86.13 99.62 102.67 106.38
3.21 3.41 2.96 4.21 2.38 2.08 2.97 4.15 4.30 5.02 2.60 2.53 4.22 6.60 4.11 3.50 3.83 3.99 3.46 3.11 2.62
3.4.5. Stability Results from our stability study showed that roemerine was very stable in all biological matrices after being stored at room temperature for 4 h, in the autosampler for 24 h, or at −20 ◦ C for 20 days (Table 3). Roemerine also showed excellent stability after three freeze–thaw cycles. 3.4.6. Dilution integrity The concentration of roemerine in liver and lung tissue homogenates exceeded the upper limit of detection. Therefore, these samples were diluted by four-fold in the same biological matrices prior to LC–MS/MS analysis. We determined the accuracy and precision of roemerine detection using diluted biological samples. The accuracy was within ±4.76% and ±7.76%, and the precision was within 5.15% and 6.8% for samples diluted in liver and lung matrices, respectively. These results indicated that the concentration of roemerine in liver and lung tissue homogenates could be determined with acceptable precision and accuracy using diluted samples. 3.4.7. Carryover evaluation Carryover for roemerine was not detectable with the chosen settings, therefore, sample carryover was not a problem in this method. 3.5. Pharmacokinetic results The validated LC–MS/MS method was successfully used to determine the concentration of roemerine in rat plasma following a single oral (20 mg/kg) or i.v. (6 mg/kg) administration. Fig. 3 shows the plasma concentration of roemerine at specific time points after administration. The pharmacokinetic parameters obtained are listed in Table 4. The rate of elimination is highly linked to the high total body clearance (CL). According to the equation Ebody = body clearance/cardiac output, CL cannot be higher than the cardiac output, and E should be considered high if above 0.35, medium if around 0.15, and low if around 0.05 [24]. In this study, CL was about 3.76 L/h/kg (Ebody ≈ 0.26) after the i.v. administration showed that roemerine was eliminated quickly in vivo. After oral administration, roemerine was rapidly absorbed and reached its peak concentration (tmax ) at approximately 0.22 h.The pharmacokinetic curve of roemerine displayed significant bimodal characteristics, which might be attributed to enterohepatic circulation, varying
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Table 3 Stability of Roemerine in plasma and tissue homogenates of rats (n = 3). Biosamples Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
QC conc (ng/mL)
Short-term stability
Postpreparative stability
Freeze–thaw stability
Long-term stability
20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
21.05 ± 0.45 207.32 ± 9.14 1523.83 ± 44.47 20.54 ± 1.06 198.50 ± 3.53 1510.38 ± 28.74 20.02 ± 0.85 196.47 ± 7.86 1546.07 ± 44.84 20.96 ± 0.11 202.91 ± 7.09 1575.82 ± 61.51 20.16 ± 0.43 200.35 ± 12.76 1498.99 ± 89.81 20.70 ± 0.76 203.66 ± 6.51 1599.10 ± 65.86 21.32 ± 0.49 205.99 ± 6.50 1528.97 ± 49.51
20.19 ± 1.36 205.44 ± 11.58 1581.34 ± 103.16 21.42 ± 0.38 211.60 ± 7.94 1618.37 ± 32.18 21.37 ± 0.40 205.02 ± 6.41 1589.93 ± 44.23 19.43 ± 0.51 204.16 ± 4.58 1515.53 ± 27.74 21.41 ± 0.32 211.79 ± 1.87 1639.27 ± 73.31 21.08 ± 0.59 207.99 ± 5.32 1636.60 ± 72.82 19.58 ± 1.02 206.13 ± 8.23 1541.60 ± 82.55
21.23 ± 0.36 212.27 ± 2.51 1544.50 ± 45.34 19.99 ± 1.08 190.26 ± 9.80 1441.73 ± 26.38 19.57 ± 0.99 196.09 ± 12.56 1506.77 ± 62.07 20.34 ± 0.60 202.04 ± 7.90 1508.21 ± 38.51 19.73 ± 2.06 196.09 ± 12.56 1473.44 ± 13.36 19.62 ± 1.72 205.15 ± 9.15 1529.75 ± 59.71 19.85 ± 1.11 204.01 ± 3.94 1487.74 ± 36.78
19.87 ± 1.76 207.64 ± 5.37 1635.26 ± 106.39 17.79 ± 0.28 182.49 ± 7.57 1451.23 ± 23.31 20.32 ± 0.74 206.26 ± 7.22 1528.02 ± 47.54 18.99 ± 0.73 183.66 ± 1.65 1445.73 ± 39.98 19.97 ± 1.03 202.03 ± 7.52 1518.22 ± 76.29 21.00 ± 0.35 196.16 ± 5.03 1518.08 ± 61.56 19.87 ± 0.99 204.08 ± 3.86 1579.04 ± 84.97
Fig. 3. Mean plasma concentration–time curves of roemerine after oral administration at doses of 20 mg/kg or i.v. administration at dose of 6 mg/kg.
absorption at different sites of the gut, or rapid tissue distribution followed by release back into the circulation. Roemerine had a t1/2 of 1.77 ± 0.41 h following i.v. administration at 6 mg/kg, with an AUC0-∞ of 1631 ± 227 and a Cmax of 1835 ± 214 g/L × h. The oral absolute bioavailability (F) of roemerine was calculated to be 84% based on the AUC0-∞ values obtained after i.v. and oral administration, indicating excellent absorption following oral administration in rats.
oral administration, with the peak level reached at 20 min in all tissues except liver. Roemerine was essentially cleared from all of the tissues at 8 h after administration. Specifically, the kinetics of roemerine tissue levels followed that of its plasma concentration. The highest tissue concentrations were found in the liver and lung, followed by kidney, spleen, and heart, in that order, which implied that the liver and lung might be the target organs of roemerine. In addition, roemerine was also detected in the brain, suggesting that roemerine is able to cross the blood–brain barrier. These results provide useful information for further studies on the mechanisms of action and toxicity of roemerine. To date there have been few pharmacokinetic or tissue distribution studies of aporphine alkaloids. We were able to find such information for five aporphine alkaloids only, which included boldine, dicentrine, magnoflorine, apomorphine, and isocorydine. These five aporphine alkaloids have the same core structure as roemerine, and the only differences are found in the substituents on the aromatic rings. Jiménez and Speisky [25] reported that boldine was rapidly (within 30 min) absorbed after oral administration and quickly eliminated following oral or intravenous (i.v.) administration in rats. Boldine was mainly distributed in the liver, also could be found in brain. Tsai et al. [26] studied the pharmacokinetics of dicentrine in rats using HPLC detection, and reported a t1/2,
3.6. Tissue distribution The concentrations of roemerine in the liver, heart, spleen, lung, kidney, and brain are shown in Fig. 4. Roemerine was detected at significant levels in all of these tissues at 5 min after Table 4 Mean pharmacokinetic parameters of roemerine in rats after oral 20 mg/kg or intravenous 6 mg/kg administration (n = 6 mean ± SD). Parameters
Oral
Intravenous
AUC0-∞ (g/L*h) t1/2 (h) CL (L/h/kg) V (L/kg) Cmax (g/L) Tmax (h) MRT0-∞ (h) F
4541 ± 464 1.59 ± 0.46 4.44 ± 0.42 10.16 ± 2.95 1358 ± 154 0.22 ± 0.084 2.83 ± 0.45 84%
1631 ± 227 1.77 ± 0.41 3.76 ± 0.63 9.39 ± 1.47 1835 ± 214 0 2.24 ± 0.39 —
Fig. 4. Mean concentrations (mean ± SD., n = 6) of roemerine in tissues at different time points after oral administration of 20 mg/kg to rats.
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of 45.20 ± 7.85 min and a CL of 0.51 ± 0.046 L/kg/min following i.v. administration, which indicated that dicentrine was also rapidly cleared. Yang et al. [27] examined the pharmacokinetics of apomorphine in beagle dogs using LC–ESI–MS detection, and reported that apomorphine was rapidly absorbed and then quickly eliminated following oral administration. Guo et al. [28] reported that isocorydine was rapidly absorbed with a tmax of 0.278 ± 0.113 h and quickly eliminated with a t1/2 of 0.906 ± 0.222 h after oral administration in rats. Moreover, isocorydine was detected in a wide range of tissue types including the brain. Zhou et al. [29] studied the pharmacokinetics of magnoflorine in rats using a new HF–LPME method, and reported a t1/2, of 3.305 ± 0.365 h and a CL of 0.07 ± 0.005 L/kg/h after i.v. administration. Considering the results from the present study, aporphine alkaloids seem to share the common pharmacokinetic characteristics of rapid absorption and elimination, although the pharmacokinetic parameters vary to a certain degree depending on the specific substituents on the aromatic rings. Similar to roemerine, all of the three aporphine alkaloids whose tissue distribution data have been reported are able to cross the blood–brain barrier. Roemerine and the other three aporphine alkaloids are all lipophilic compounds, they can cross the blood–brain barrier by passive diffusion, and they are able to cross the blood–brain barrier may be due to the high lipophilicity and probable weak Pgp substrate. 4. Conclusions In this study, we developed and validated a new LC–MS/MS method for the detection of roemerine in rat plasma and tissue samples and successfully used it to study the pharmacokinetics and tissue distribution of roemerine in rats. To the best of our knowledge, this is the first report on the pharmacokinetics and tissue distribution of roemerine in vivo. A tmax of approximately 0.22 h was observed following oral administration roemerine indicating rapid absorption in rats, and a relatively short t1/2 of less than 1.77 h and a high CL of 4.44 L/h/kg were observed also indicating rapid elimination. The absolute bioavailability of roemerine was determined to be 84% following oral administration. Furthermore, we found that roemerine was rapidly and widely distributed in various tissues. The findings of this study provide critical information for potential clinical studies of roemerine in the future. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81173643) and the Science Research Foundations of Yunnan Province (No.2010CD114).
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Plasma pharmacokinetics and tissue distribution study of roemerine in rats by liquid chromatography with tandem mass spectrometry (LC–MS/MS) Yue-Qiong Liu a,b , Gong-Hao He a , Hong-Liang Li c , Jiang-Chang He a , En-Fu Feng a , Lan Bai a , Cheng-Ying Wang a , Gui-Li Xu a,∗ a
Department of Pharmacy, Kunming General Hospital of Chengdu Military Region, Kunming 650032, PR China Department of Pharmacy, Wenshan city People’ Hospital, Wenshan 663000, PR China c Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China b
a r t i c l e
i n f o
Article history: Received 10 June 2014 Received in revised form 22 August 2014 Accepted 25 August 2014 Available online 28 August 2014 Keywords: Roemerine Pharmacokinetics Tissue distribution LC–MS/MS
a b s t r a c t In the present study, a new LC–MS/MS method for the determination of roemerine in rat plasma and tissue samples was developed and successfully used to study the pharmacokinetics and tissue distribution of roemerine after oral and intravenous (i.v.) administration in rats. The plasma and tissue samples were processed by liquid–liquid extraction with n-hexane. Isocorydine was used as the internal standard (IS) for sample processing and analysis. The MS/MS detection was carried out by monitoring the transitions of m/z 280→249 and m/z 342→279 for roemerine and the IS, respectively. The calibration curve displayed excellent linearity over the concentration range of 10–2000 ng/mL (n = 8, r2 ≥ 0.995), and the lower limit of quantification (LLOQ) was determined to be 10 ng/mL. This method was rapid, accurate, highly sensitive, and fully validated. The pharmacokinetic study showed that roemerine was rapidly absorbed and eliminated with a tmax of 0.22 ± 0.08 h, t1/2 of 1.59 ± 0.46 h, CL of 4.44 ± 0.42 L/h/kg, and Vd of 10.16 ± 2.95 g/L following oral administration. Additionally, roemerine showed an excellent oral bioavailability of 84% and a wide tissue distribution with brain penetration. Highest concentrations of roemerine were found in the liver and lung, followed by kidney, spleen, heart, and brain, in that order. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Roemerine (Fig. 1A) is an aporphine alkaloid found in many plants such as Nelumbo nucifera [1,2], Stephania kwangsiensis [3], Turkish Papaver [4], Rollinia leptopetala [5], Hornschuchia oblique [6], and Neolitsea dealbata [7]. Roemerine has also been produced by biosynthesis [8]. Pharmacological studies have shown that roemerine possesses diverse beneficial bioactivities. It is a vasodilator that inhibits the noradrenaline-induced contraction [9,10]. It also interacts with the P-glycoprotein and enhances the cytotoxicity of vinblastine against multidrug-resistant KB-V1 cells [11]. In addition, it binds to the 5-HT2A receptor with high affinity, which suggests that it may have CNS effects [12,13]. Roemerine also exhibits antiplasmodial [14], antibacterial [15], antifungal [16], and anthelmintic [17] activities. Moreover, roemerine regulates glucose metabolism by stimulating glucose consumption and inhibiting glucose absorption [18,19]. Therefore, roemerine may have potential value as
therapeutic agent for a variety of diseases. Nonetheless, in contrast to the comprehensive research on bioactivity, to date the pharmacokinetic characteristics of roemerine have not been reported. Roemerine in raw plant extracts has been determined using high performance liquid chromatography (HPLC) coupled with a diode array detector [20,21]. However, this method is not suitable for pharmacokinetic studies of roemerine, which require pretreatment of samples and higher sensitivity levels of detection. LC–MS/MS is a well-established analytical method with high specificity, high sensitivity, and high throughput [22]. In the present work, we first developed and validated a new LC–MS/MS method for determination of roemerine in rat plasma and tissue samples. This method was successfully used to study the pharmacokinetics, oral bioavailability, and tissue distribution of roemerine in rats. 2. Experimental 2.1. Chemicals and reagents
∗ Corresponding author. Tel./fax: +86 871 4774784. E-mail address: [email protected] (G.-L. Xu). http://dx.doi.org/10.1016/j.jchromb.2014.08.031 1570-0232/© 2014 Elsevier B.V. All rights reserved.
Roemerine (purity 97%) and isocorydine (purity 98%; Fig. 1B) were kindly provided by Professor Gao-Xiong Rao of the School
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Fig. 1. Chemical structures and product ion spectra of roemerine (A) and isocorydine (B) in positive mode.
of Pharmacy, Yunnan College of Traditional Chinese Medicine. Methanol, acetic acid, and n-hexane (all HPLC grade) were purchased from Merck KGaAS (Darmstadt, Germany). Distilled, deionized water freshly generated on a Milli-Q reagent water system (Millipore, USA) was used in all sample preparations and chromatography. All other reagents were of analytical grade and purchased from commercial sources. 2.2. LC–MS/MS instrument and conditions The HPLC was performed on an Agilent 1200 series HPLC system (Agilent, USA) equipped with an autosampler, a degasser, and two SL bin-pumps. The chromatographic separation was achieved on a symmetry C18 analytical column (50 mm × 2.1 mm, 5 m) connected to a SecurityGuard Cartridge AQC18 guard column (30 mm × 4 mm, 5 m), thermostated at 35 ◦ C. The samples (10 L each) were run using an isocratic mobile phase of methanol–water mixture (50:50, v/v) containing 0.1% acetic acid, at a flow rate of 0.2 mL/min. The Agilent 1200 HPLC system was coupled on-line to a G6410B triple quadruple mass spectrometer (Agilent, USA) equipped with an electrospray ionization (ESI) source. The mass spectrometer was operated using the ESI positive ion mode under multiplereaction monitoring (MRM). The optimized precursor-to-product ion transitions were monitored at m/z 280→249 and m/z 342→279 for roemerine and the IS, respectively. The optimized parameters for MS analysis were: capillary voltage 4000 V; nebulizer pressure 35 psi; dry gas temperature 350 ◦ C; gas flow 10 L/min; collision energies (CE) 13 eV; fragmentor potential 78 V and 86 V for roemerine and the IS, respectively. 2.3. Standard and quality control (QC) sample preparation The stock solutions of roemerine and IS were both prepared in methanol at a final concentration of 1 mg/mL. Working solutions
of roemerine (100–20000 ng/mL) and the IS (2000 ng/mL) were obtained by diluting the stock solutions with methanol–H2 O (50:50, v/v). All of the solutions were kept at −4 ◦ C until analysis. The calibration standards of roemerine (10–2000 ng/mL) were prepared by adding 10 L of the working solution at different concentrations to 90 L of the blank biological matrix. QC samples of roemerine (20, 200, and 1600 ng/mL) were similarly prepared. 2.4. Sample preparation In the present study, roemerine and the IS were extracted from rat plasma and tissue homogenates using a simple liquid–liquid extraction (LLE) method. After samples were thawed at room temperature, an aliquot of 100 L plasma or tissue homogenates was transferred into a 2 mL tube. The sample was first vortex-mixed with 10 L of IS working solution and then with 1 mL of n-hexane for extraction. After vortex for 2 min and centrifuged at 10,000 rpm for 5 min, the supernatant was transferred into another tube and evaporated to dryness under a gentle stream of nitrogen at 25 ◦ C. The residue was reconstituted with 100 L methanol–H2 O (50:50, v/v) and subjected to LC–MS/MS analysis. 2.5. Method validation The developed method was validated according to the FDA guidelines for the validation of bioanalytical methods [23]. 2.5.1. Specificity Specificity of the method was assessed by analyzing blank biological samples from six different sources, blank biological samples spiked with roemerine and the IS, and plasma and tissue samples collected after oral administration of roemerine, which were also spiked with the IS.
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2.5.2. Linearity and LLOQ The calibration curves were generated by plotting the peak area ratios of roemerine to the IS (y-axis) versus nominal concentrations of roemerine (x-axis) using a weighted linear regression (1/x2 ). The LLOQ was defined as the lowest concentration of roemerine in the calibration curve that could be detected with an acceptable precision not exceeding 20% and an accuracy within ±20%. 2.5.3. Accuracy and precision The intraday accuracy and precision were determined by analyzing six replicates of the low, medium, and high QC samples (20, 200, and 1600 ng/mL, respectively). The interday accuracy and precision were determined over test results in three consecutive days. An accuracy within ±15% of the relative error (RE) from the nominal values and a precision ≤15% of the relative standard deviation (RSD) were deemed to be acceptable. 2.5.4. Recovery and matrix effect The extraction recovery in rat sample matrices for roemerine and the IS was calculated as the peak area ratios of the rat sample matrix spiked with a standard solution to the blank matrix spiked with an equivalent standard solution. The recovery of roemerine was determined at low, medium and high concentrations, while the recovery of the IS was determined at a single concentration of 200 ng/mL. The matrix effect of extraction on roemerine analysis was evaluated by comparing the peak areas of the n-hexane extracted blank samples spiked with roemerine at three QC concentrations with those of roemerine standard solutions at equivalent concentrations. The matrix effect of extraction on IS analysis was similarly evaluated. 2.5.5. Stability The stability of roemerine in rat biological samples was studied using blank biological samples spiked with QC solutions at low, medium, and high concentrations in triplicate. The stability of samples was analyzed under four different conditions: (1) after 20 days of storage at −20 ◦ C (long-term stability); (2) after 4 h at room temperature (short-term stability); (3) after three freeze–thaw (−20 ◦ C/room temperature) cycles; and (4) after 24 h in the autosampler (post-preparation stability). The stability was deemed acceptable when the percentage deviation was within ±15%. 2.5.6. Dilution integrity The blank liver and lung samples spiked with 6400 ng/mL of roemerine (exceeding the upper limit of the calibration curve) were diluted by four-fold and subjected to LC–MS/MS analysis. The experiments were conducted in six replicates. The dilution integrity was deemed acceptable when the precision and accuracy were within ±15%. 2.5.7. Carryover evaluation Carryover of roemerine was assessed by injection of blank samples (no roemerine and the IS) directly after injection of the highest point in the calibration curve (2000 ng/mL). Carry over in the blank sample following the high concentration standard should not be greater than 20% of the LLOQ and 5% for the internal standard. 2.6. Animals The animal study protocol was approved by the Institutional Animal Care and Use Committee of Kunming General Hospital of Chengdu Military Region. Sprague–Dawley rats (200 ± 20 g, both male and female) were purchased from the Central Animal House of Kunming Medical University and acclimated in the laboratory for
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1 week prior to the experiments. All of the rats were housed and cared for under constant temperature of 23 ± 1 ◦ C and humidity of 60 ± 10% with free access to food and water. Rats were fasted for 12 h with free access to water prior to roemerine administration. 2.7. Pharmacokinetic study For pharmacokinetic studies, the rats were randomly divided into two groups (n = 6 for each group). Roemerine [injectable solution in ethanol/Tween 80/0.9% NaCl (1:0.2:8.8, v/v/v)] was administered at an oral dose of 20 mg/kg or an intravenous dose of 6 mg/kg. At specific time points after administration (oral: 5 min to 24 h; i.v.: 0 min to 12 h), blood samples (500 L) were collected from the epicanthic veins of rats into heparinized tubes. Samples were immediately centrifuged at 10,000 rpm for 5 min and the supernatant plasma samples were collected and stored at −20 ◦ C until analysis. 2.8. Tissue distribution study The rats were randomly divided into five groups (n = 6 for each group). The rats were sacrificed at 5 min, 20 min, 45 min, 2 h, and 8 h, respectively, after oral administration of roemerine (20 mg/kg). The liver, heart, spleen, lung, kidney, and brain tissues were collected immediately, rinsed with physiological saline solution, blotted dry with filter paper, and homogenized in physiological saline solution (1:2, w/v) with Superfine Homogenizer. The tissue homogenates were centrifuged and aliquots of the supernatants were collected and stored at −20 ◦ C until analysis. 2.9. Data analysis The plasma and tissue homogenates were subjected to LC–MS/MS analysis as above and the concentrations of roemerine were calculated according to the calibration curves. The pharmacokinetic parameters including the area under the curve (AUC), half-life (t1/2 ), apparent central volume of distribution (V), and clearance rate (CL) were calculated by the DAS 3.0 software (Drug and Statistics, Mathematical Pharmacology Professional Committee of China, Shanghai, China) with a noncompartmental model. The maximum plasma concentration (Cmax ) and the time to reach Cmax (tmax ) were calculated from the experimental data. Absolute bioavailability (F) was calculated based on the AUC0-∞ of i.v. and oral administration at equivalent doses. All data are presented as means ± standard deviation (SD). 3. Results and discussion 3.1. LC–MS/MS conditions In order to identify the optimal conditions for the determination of roemerine and the IS, we investigated a variety of mobile phase compositions and elution flow rates. We tested mobile phases with varying organic solvent/water compositions and different pH modifiers, the test results showed that the optimal retention time and ionization of roemerine and IS were obtained using the mobile phase of methanol–water (50:50, v/v) containing 0.1% acetic acid. Moreover, we tested elusion flow rate in the range of 0.15–0.4 mL/min, and the flow rate at 0.2 mL/min was selected for sample analysis based on performance. Under the optimized conditions, we did not detect any significant endogenous interference. We tested the positive- and negative-ion ESI modes for determination of roemerine in biological samples. In the positive mode, roemerine showed a strong response of protonated molecular ions [M + H]+ at m/z 280, the signal to noise ratio (s/n) is far higher than
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Fig. 2. Representative chromatograms of blank plasma (A) and blank liver homogenates (B), blank plasma spiked with roemerine (10 ng/mL) and IS (200 ng/mL) (C) and blank liver homogenates spiked with roemerine (10 ng/mL) and IS (200 ng/mL) (D), real plasma sample (491.14 ng/mL) (E) and real liver homogenates (742.93 ng/mL) (F) after oral administration of 20 mg/kg roemerine spiked with IS.
that in negative ions. In addition, the fragmentation in ESI+ is usually richer than that in ESI− . Therefore, the ESI positive ion mode was used for subsequent sample analysis. The collision induced dissociation (CID) parameters were optimized under the MRM mode for stable ion response and high detection sensitivity. Further, the most suitable conditions for mass spectrometry were determined by optimizing collision energy, fragmentor potential, source temperature, drying gas temperature, drying gas flow, nebulizer pressure, and capillary voltage. Under the optimized conditions, the peaks with the highest intensity in the MS spectra of roemerine and the IS were the ions at m/z 280 [M + H]+ and m/z 342 [M + H]+ , respectively. After fragmentation in the collision cell, the most abundant and stable product ions were at m/z 249 and m/z 279 (Fig. 1), respectively.
20% for roemerine, whilst the SPE method suffered a low recovery (approximately 65%). The LLE method demonstrated the best performance among the three methods, and was thus selected as the method for sample pretreatment. We subsequently tested several organic solvents including ethyl acetate, dichloromethane, methyl t-butyl ether, and n-hexane as the extraction solvent in the LLE method for plasma and tissue sample pretreatment. We found that the best overall result (high recovery, low matrix suppression, and fast solvent evaporation) was obtained when n-hexane was used as the extraction solvent. The LLE sample pretreatment with nhexane was quick, convenient, and effective, and only required small amounts of solvent and biological matrix.
3.2. Selection of internal standard
3.4.1. Specificity Fig. 2 shows representative chromatograms of blank matrices, blank matrices spiked with roemerine and the IS, and plasma and tissue samples collected after oral administration of roemerine, which were also spiked with the IS. The retention times of roemerine and the IS were approximately 0.9 min and 0.6 min, respectively. The peaks were well separated with no detectable interfering peaks nearby when blank plasma or tissue homogenate samples spiked with roemerine and the IS were analyzed.
For a proper internal standard, a similar chemical behavior and a suitable retention time were of significant importance. In addition, an analog has to be used because labeled roemerine was not commercially available. Isocorydine, an analog to roemerine, was chosen as the IS because it has similar chemical structure, chromatographic behavior, and MS characteristics to roemerine.
3.4. Method validation
3.3. Optimization of sample pretreatment We tested sample preparation by LLE, solid-phase extraction (SPE), and protein precipitation (PPT). The PPT method showed severe matrix suppression, with a matrix effect of approximately
3.4.2. Linearity and LLOQ Calibration curves were established over the concentration range between 10 and 2000 ng/mL for roemerine in all biological matrices. The calibration curves in all biological matrices
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Table 1 Precision and accuracy of roemerine in plasma and tissue homogenates of rats (n = 6). Biosamples
QC conc (ng/mL) 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
Precision (RSD, %) Intra-day
Inter-day
Accuracy (RE, %) Intra-day
Inter-day
4.36 3.05 1.08 1.82 5.12 3.50 2.52 4.56 3.51 5.12 4.20 3.36 2.89 5.58 4.61 3.97 3.21 4.54 4.33 1.48 3.03
4.23 5.31 2.13 5.82 3.46 5.60 3.65 2.74 3.89 2.65 2.71 2.06 4.02 4.86 3.46 3.43 6.46 1.74 1.03 4.59 0.99
–0.85 0.61 –6.50 6.95 0.50 –5.97 4.87 1.29 –5.64 4.61 –1.07 –5.49 2.60 –0.65 –7.63 2.41 –7.96 –5.46 3.00 6.10 –5.70
2.29 0.51 –5.60 1.28 –1.68 –6.41 2.84 1.87 –5.91 2.13 0.61 –3.24 2.15 –0.25 –6.90 5.90 –0.62 –4.04 1.89 1.25 –5.35
exhibited excellent linearity (r2 ≥ 0.995). The LLOQ of roemerine was determined to be 10 ng/mL (S/N ≥ 10) in all biological matrices, the precision and accuracy of LLOQ were less than 10.55% and within ± 9.09%. 3.4.3. Accuracy and precision The intra- and inter-day accuracy and precision for roemerine in biological samples are summarized in Table 1. The relative errors (RE, %) of assay accuracy ranged from −7.96 to 6.95%, and the precision values (RSD, %) were within 6.64%. These results were all within the threshold of ±15%, which indicated that the method had acceptable accuracy, precision, and reproducibility. 3.4.4. Recovery and matrix effect The extraction recovery and matrix effect for roemerine in plasma and tissue matrices are shown in Table 2. These data indicated reproducible and acceptable recoveries in all biological matrices with no significant ion suppression or enhancement effects. Table 2 Matrix effect and recovery of roemerine in plasma and tissue homogenates of rats (n = 6). Biosamples
Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
QC conc (ng/mL)
20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
Recovery (%)
Matrix effect (%)
Mean
RSD
Mean
RSD
85.22 89.42 87.57 82.60 83.55 85.55 82.55 84.88 80.13 82.18 84.15 82.98 86.67 84.37 84.03 70.88 69.05 69.33 78.33 76.52 75.82
2.56 2.53 3.58 4.42 3.32 4.74 4.38 4.94 3.76 4.84 3.55 2.87 2.32 2.69 3.49 2.62 5.23 5.49 4.70 3.79 3.43
90.53 88.06 86.17 100.27 105.45 107.45 99.35 90.62 92.65 100.05 98.32 104.17 100.57 87.95 93.28 85.1 91.48 86.13 99.62 102.67 106.38
3.21 3.41 2.96 4.21 2.38 2.08 2.97 4.15 4.30 5.02 2.60 2.53 4.22 6.60 4.11 3.50 3.83 3.99 3.46 3.11 2.62
3.4.5. Stability Results from our stability study showed that roemerine was very stable in all biological matrices after being stored at room temperature for 4 h, in the autosampler for 24 h, or at −20 ◦ C for 20 days (Table 3). Roemerine also showed excellent stability after three freeze–thaw cycles. 3.4.6. Dilution integrity The concentration of roemerine in liver and lung tissue homogenates exceeded the upper limit of detection. Therefore, these samples were diluted by four-fold in the same biological matrices prior to LC–MS/MS analysis. We determined the accuracy and precision of roemerine detection using diluted biological samples. The accuracy was within ±4.76% and ±7.76%, and the precision was within 5.15% and 6.8% for samples diluted in liver and lung matrices, respectively. These results indicated that the concentration of roemerine in liver and lung tissue homogenates could be determined with acceptable precision and accuracy using diluted samples. 3.4.7. Carryover evaluation Carryover for roemerine was not detectable with the chosen settings, therefore, sample carryover was not a problem in this method. 3.5. Pharmacokinetic results The validated LC–MS/MS method was successfully used to determine the concentration of roemerine in rat plasma following a single oral (20 mg/kg) or i.v. (6 mg/kg) administration. Fig. 3 shows the plasma concentration of roemerine at specific time points after administration. The pharmacokinetic parameters obtained are listed in Table 4. The rate of elimination is highly linked to the high total body clearance (CL). According to the equation Ebody = body clearance/cardiac output, CL cannot be higher than the cardiac output, and E should be considered high if above 0.35, medium if around 0.15, and low if around 0.05 [24]. In this study, CL was about 3.76 L/h/kg (Ebody ≈ 0.26) after the i.v. administration showed that roemerine was eliminated quickly in vivo. After oral administration, roemerine was rapidly absorbed and reached its peak concentration (tmax ) at approximately 0.22 h.The pharmacokinetic curve of roemerine displayed significant bimodal characteristics, which might be attributed to enterohepatic circulation, varying
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Table 3 Stability of Roemerine in plasma and tissue homogenates of rats (n = 3). Biosamples Plasma Liver
Heart
Spleen
Lung
Kidney
Brain
QC conc (ng/mL)
Short-term stability
Postpreparative stability
Freeze–thaw stability
Long-term stability
20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600 20 200 1600
21.05 ± 0.45 207.32 ± 9.14 1523.83 ± 44.47 20.54 ± 1.06 198.50 ± 3.53 1510.38 ± 28.74 20.02 ± 0.85 196.47 ± 7.86 1546.07 ± 44.84 20.96 ± 0.11 202.91 ± 7.09 1575.82 ± 61.51 20.16 ± 0.43 200.35 ± 12.76 1498.99 ± 89.81 20.70 ± 0.76 203.66 ± 6.51 1599.10 ± 65.86 21.32 ± 0.49 205.99 ± 6.50 1528.97 ± 49.51
20.19 ± 1.36 205.44 ± 11.58 1581.34 ± 103.16 21.42 ± 0.38 211.60 ± 7.94 1618.37 ± 32.18 21.37 ± 0.40 205.02 ± 6.41 1589.93 ± 44.23 19.43 ± 0.51 204.16 ± 4.58 1515.53 ± 27.74 21.41 ± 0.32 211.79 ± 1.87 1639.27 ± 73.31 21.08 ± 0.59 207.99 ± 5.32 1636.60 ± 72.82 19.58 ± 1.02 206.13 ± 8.23 1541.60 ± 82.55
21.23 ± 0.36 212.27 ± 2.51 1544.50 ± 45.34 19.99 ± 1.08 190.26 ± 9.80 1441.73 ± 26.38 19.57 ± 0.99 196.09 ± 12.56 1506.77 ± 62.07 20.34 ± 0.60 202.04 ± 7.90 1508.21 ± 38.51 19.73 ± 2.06 196.09 ± 12.56 1473.44 ± 13.36 19.62 ± 1.72 205.15 ± 9.15 1529.75 ± 59.71 19.85 ± 1.11 204.01 ± 3.94 1487.74 ± 36.78
19.87 ± 1.76 207.64 ± 5.37 1635.26 ± 106.39 17.79 ± 0.28 182.49 ± 7.57 1451.23 ± 23.31 20.32 ± 0.74 206.26 ± 7.22 1528.02 ± 47.54 18.99 ± 0.73 183.66 ± 1.65 1445.73 ± 39.98 19.97 ± 1.03 202.03 ± 7.52 1518.22 ± 76.29 21.00 ± 0.35 196.16 ± 5.03 1518.08 ± 61.56 19.87 ± 0.99 204.08 ± 3.86 1579.04 ± 84.97
Fig. 3. Mean plasma concentration–time curves of roemerine after oral administration at doses of 20 mg/kg or i.v. administration at dose of 6 mg/kg.
absorption at different sites of the gut, or rapid tissue distribution followed by release back into the circulation. Roemerine had a t1/2 of 1.77 ± 0.41 h following i.v. administration at 6 mg/kg, with an AUC0-∞ of 1631 ± 227 and a Cmax of 1835 ± 214 g/L × h. The oral absolute bioavailability (F) of roemerine was calculated to be 84% based on the AUC0-∞ values obtained after i.v. and oral administration, indicating excellent absorption following oral administration in rats.
oral administration, with the peak level reached at 20 min in all tissues except liver. Roemerine was essentially cleared from all of the tissues at 8 h after administration. Specifically, the kinetics of roemerine tissue levels followed that of its plasma concentration. The highest tissue concentrations were found in the liver and lung, followed by kidney, spleen, and heart, in that order, which implied that the liver and lung might be the target organs of roemerine. In addition, roemerine was also detected in the brain, suggesting that roemerine is able to cross the blood–brain barrier. These results provide useful information for further studies on the mechanisms of action and toxicity of roemerine. To date there have been few pharmacokinetic or tissue distribution studies of aporphine alkaloids. We were able to find such information for five aporphine alkaloids only, which included boldine, dicentrine, magnoflorine, apomorphine, and isocorydine. These five aporphine alkaloids have the same core structure as roemerine, and the only differences are found in the substituents on the aromatic rings. Jiménez and Speisky [25] reported that boldine was rapidly (within 30 min) absorbed after oral administration and quickly eliminated following oral or intravenous (i.v.) administration in rats. Boldine was mainly distributed in the liver, also could be found in brain. Tsai et al. [26] studied the pharmacokinetics of dicentrine in rats using HPLC detection, and reported a t1/2,
3.6. Tissue distribution The concentrations of roemerine in the liver, heart, spleen, lung, kidney, and brain are shown in Fig. 4. Roemerine was detected at significant levels in all of these tissues at 5 min after Table 4 Mean pharmacokinetic parameters of roemerine in rats after oral 20 mg/kg or intravenous 6 mg/kg administration (n = 6 mean ± SD). Parameters
Oral
Intravenous
AUC0-∞ (g/L*h) t1/2 (h) CL (L/h/kg) V (L/kg) Cmax (g/L) Tmax (h) MRT0-∞ (h) F
4541 ± 464 1.59 ± 0.46 4.44 ± 0.42 10.16 ± 2.95 1358 ± 154 0.22 ± 0.084 2.83 ± 0.45 84%
1631 ± 227 1.77 ± 0.41 3.76 ± 0.63 9.39 ± 1.47 1835 ± 214 0 2.24 ± 0.39 —
Fig. 4. Mean concentrations (mean ± SD., n = 6) of roemerine in tissues at different time points after oral administration of 20 mg/kg to rats.
Y.-Q. Liu et al. / J. Chromatogr. B 969 (2014) 249–255
of 45.20 ± 7.85 min and a CL of 0.51 ± 0.046 L/kg/min following i.v. administration, which indicated that dicentrine was also rapidly cleared. Yang et al. [27] examined the pharmacokinetics of apomorphine in beagle dogs using LC–ESI–MS detection, and reported that apomorphine was rapidly absorbed and then quickly eliminated following oral administration. Guo et al. [28] reported that isocorydine was rapidly absorbed with a tmax of 0.278 ± 0.113 h and quickly eliminated with a t1/2 of 0.906 ± 0.222 h after oral administration in rats. Moreover, isocorydine was detected in a wide range of tissue types including the brain. Zhou et al. [29] studied the pharmacokinetics of magnoflorine in rats using a new HF–LPME method, and reported a t1/2, of 3.305 ± 0.365 h and a CL of 0.07 ± 0.005 L/kg/h after i.v. administration. Considering the results from the present study, aporphine alkaloids seem to share the common pharmacokinetic characteristics of rapid absorption and elimination, although the pharmacokinetic parameters vary to a certain degree depending on the specific substituents on the aromatic rings. Similar to roemerine, all of the three aporphine alkaloids whose tissue distribution data have been reported are able to cross the blood–brain barrier. Roemerine and the other three aporphine alkaloids are all lipophilic compounds, they can cross the blood–brain barrier by passive diffusion, and they are able to cross the blood–brain barrier may be due to the high lipophilicity and probable weak Pgp substrate. 4. Conclusions In this study, we developed and validated a new LC–MS/MS method for the detection of roemerine in rat plasma and tissue samples and successfully used it to study the pharmacokinetics and tissue distribution of roemerine in rats. To the best of our knowledge, this is the first report on the pharmacokinetics and tissue distribution of roemerine in vivo. A tmax of approximately 0.22 h was observed following oral administration roemerine indicating rapid absorption in rats, and a relatively short t1/2 of less than 1.77 h and a high CL of 4.44 L/h/kg were observed also indicating rapid elimination. The absolute bioavailability of roemerine was determined to be 84% following oral administration. Furthermore, we found that roemerine was rapidly and widely distributed in various tissues. The findings of this study provide critical information for potential clinical studies of roemerine in the future. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81173643) and the Science Research Foundations of Yunnan Province (No.2010CD114).
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