Journal of Chromatography B, 961 (2014) 20–28
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
A sensitive liquid chromatography–tandem mass spectrometry method for pharmacokinetics and tissue distribution of nuciferine in rats Shengying Gu, Guanhua Zhu, Yuzhu Wang, Qin Li, Xin Wu, Jigang Zhang, Gaolin Liu ∗ , Xiaoyu Li ∗ Department of Clinic Pharmacy, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 24 February 2014 Accepted 22 April 2014 Available online 5 May 2014 Keywords: Nuciferine LC–MS/MS Pharmacokinetics Tissue distribution
a b s t r a c t Nuciferine is an important drug candidate for the treatment of obesity-related diseases. However, few investigations have been conducted about the pharmacokinetics and tissue distribution of nuciferine to better understand its behavior and action mechanism in vivo. Thus, a sensitive and reliable liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) method was established and validated for the quantification of nuciferine in rat plasma and tissue samples. The validated method was successfully applied to the pharmacokinetic and tissue distribution study of nuciferine in rats. One-compartmental pharmacokinetic parameters indicated that nuciferine had rapid distribution, extensive tissue uptake, and poor absorption into systemic circulation. The values of absolute bioavailability were (3.8 ± 1.4)%, (4.2 ± 1.3)% and (3.9 ± 1.0)% after oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine and intravenous administration of 0.2 mg/kg nuciferine in rats. The results of the tissue distribution study suggested that nuciferine was distributed into the brain, liver and adipose tissue after intravenous administration. In conclusion, the present study may provide a material basis for study of the pharmacological action of nuciferine in the treatment of obesity, and meaningful insights into further study on dosage modification. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lotus (Nelumbo nucifera Gaertn) leaf is a traditional Chinese medicine used for clearing heat, removing heatstroke, cooling and stanching blood flow [1,2]. Recent pharmacological studies have demonstrated that lotus leaf also has an anti-obesity effect by inhibiting the absorption of lipids and carbohydrates, accelerating lipid metabolism [3,4], and reducing triglyceride accumulation in adipocytes [5]. This effect is supposed to be attributed to alkaloid nuciferine, the major constituent extracted from lotus leaf [6]. Nuciferine ((R)-1, 2-dimethoxyaporphine) is an aromatic ethercontaining compound whose chemical structure is shown in Fig. 1. Previous studies reported that nuciferine possessed anti-melanogenesis [7], anti-HIV [8], anti-poliovirus [9], antiacetylcholinesterase [10] and insulin secretagogue activities [11]. Recent studies have demonstrated that nuciferine can ameliorate
∗ Corresponding authors at: 650# New Songjiang Road, Songjiang District, Shanghai 200080, People’s Republic of China. Tel.: +86 21 37798312. E-mail addresses: [email protected] (G. Liu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.jchromb.2014.04.038 1570-0232/© 2014 Elsevier B.V. All rights reserved.
hyperlipemia, lower cholesterols and improve hepatic lipid metabolism [12,13]. Given these remarkable functions, more attention has been paid to the pharmacological research on nuciferine and its clinical therapeutic application. Pharmacokinetics and characteristics of tissue distribution are vital to the understanding of the in vivo behavior and action mechanism. So there is an increasing demand on quality control of nuciferine. Several techniques have been employed for analysis of nuciferine, such as thin-layer chromatography (TLC) [14], high-performance liquid chromatography (HPLC) with ultraviolet (UV) [15], HPLC coupled with mass spectrometry (MS) [16] and non-aqueous capillary electrophoresis using UV/MS [17]. However, most of these methods are time-consuming and lack complete validation data or sensitive pharmacokinetics study. Xie et al. [18] developed a HPLC–MS/MS method with the lower limit of quantification (LLOQ) of 1 ng/ml and applied it to the pharmacokinetics study of nuciferine after oral administration of 20 mg/kg in rats. However, the pharmacodynamic study by Lin et al. [19] reported that the daily intake of nuciferine was (3.2–12.8) mg/kg in rats, suggesting that the pharmacokinetics study of Xie et al. might be of little guiding significance and the sensitivity of Xie’s HPLC–MS/MS
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
21
Fig. 1. The chemical structure of nuciferine and papaverine (IS).
method could not meet the requirement of low-dose nuciferine. The goal of this study was to develop and validate a sensitive and reliable reversed phase LC–MS/MS method and applied it to study pharmacokinetics and tissue distribution of nuciferine in rats, hoping that it could provide helpful information for the development of suitable dosage forms and clinical application of nuciferine. 2. Experimental 2.1. Chemicals and materials Nuciferine (purity ≥ 98.0%) was purchased from ZR Chemicals (Shanghai, China). Papaverine as internal standard (IS, purity ≥ 99.9%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). LC–MS grade ammonium acetate was purchased from CNW Technologies GmbH (Dusseldorf, Germany). HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). HPLC grade ethylacetate and sodium carbonate anhydrous were purchased from Aladdin (China). Water was purified by employing a Milli-Q water purification system (Millipore, France). 2.2. Instrumentation and LC–MS/MS method The LC–MS/MS system consisted of a Shimadzu LC-20A chromatographic system (Shimadzu, Japan) and an API 4000 mass spectrometer (AB SCIEX, Singapore) equipped with electrospray ionization (ESI) source system. The system control and data analysis were performed by AB Sciex Analyst software (the software version: Analyst 1.5.1). Chromatographic separation was carried out on a Shim-pack XD-ODS column (50 mm × 3 mm, 2.2 m) with a Shim-pack GVDODS (5.0 mm × 2.0 mm, 2.2 m) guard column (Shimadzu, Japan). The HPLC was operated with a gradient mobile phase system consisting of water containing 5 mM ammonium acetate (phase A) and acetonitrile (phase B) at a flow rate of 0.26 ml/min. The pump was programmed as follows: phase B was increased from 15% to 60% within the first 3.5 min, held for 1 min, decreased to 15% within the next 1.5 min, and held for 0.5 min (total gradient time: 6.5 min). A 5 l sample was injected into the system with the auto-sampler conditioned at 4 ◦ C and column temperature maintained at 35 ◦ C. The mass spectrometer was operated in a positive ion mode. The multiple reaction monitoring (MRM) transitions were performed at m/z 296.1 → 265.0 for nuciferine and m/z 340.2 → 202.2 for papaverine (IS). Optimized values for declustering potential (DP), collision energy (CE), entrance potential (EP) and collision exit potential (CXP) were 80 V, 24 eV, 10 V and 15 V for nuciferine, and 90 V, 36 eV, 10 V and 15 V for IS, respectively. The MS parameters
were as follows: curtain gas of 12 psi; Ion spray voltage of 5500 V; source temperature of 500 ◦ C; Ion source Gas 1 and Gas 2 (nitrogen) of 40 psi and 50 psi. 2.3. Standard solutions, calibration and quality control samples Stock solutions were prepared by dissolving nuciferine and IS in methanol to obtain the concentrations of 1.0 and 1.2 mg/ml, respectively. Subsequently, a series of standard working solutions was prepared by diluting stock solution with methanol to get the concentrations of 0.1–100 ng/ml. A 120 ng/ml solution for IS was prepared in methanol. Calibration curves were prepared by spiking with 10 l different concentrations of the standard working stock solutions, 10 l IS solution (papaverine 120 ng/ml) and 100 l blank rat plasma or tissue homogenate sample to 1.5 ml Eppendorf tube on the analysis day. Calibration curves were prepared at concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 ng/ml for the plasma and lung; 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 ng/ml for the heart and adipose tissue; 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml for the liver, spleen, kidney and brain, respectively. The similar method was also used to prepare quality control samples (low QC, med QC and high QC) in rat plasma or tissue homogenates at concentrations of 0.25, 5 and 80 ng/ml for the plasma and lung; 0.25, 1 and 8 ng/ml for the heart and adipose tissue; 0.25, 2.5 and 40 ng/ml for the liver, spleen, kidney and brain. 2.4. Sample preparation A rat plasma or tissue homogenate sample (100 l) was placed in a 1.5 ml Eppendorf tube and mixed with 30 l 5.3% Na2 CO3 solution and 10 l IS solution (papaverine 120 ng/ml) for 3 min by vortexing. The mixture was extracted with ethyl acetate (1 ml) by vortexing and then centrifuged at 13,000 rpm for 5 min. The supernatant (900 l) was transferred to a new 1.5 ml Eppendorf tube and evaporated to dryness under vacuum. The dried residue was reconstituted with 100 l acetonitrile, vortex-mixed for 3 min and centrifuged at 13,000 rpm for 5 min. Finally, the supernatant liquid (5 l) was injected into the LC–ESI-MS/MS system. 2.5. Method validation This HPLC–MS/MS method was validated in accordance with the guidelines for Bioanalytical Method Validation published by the US FDA and other related guidelines with respect to specificity, linearity, precision and accuracy, recovery and matrix effect, stability, dilution and carry over [20,21]. Six blank rat plasma samples and tissue homogenate as a representative sample were used to evaluate the specificity. The linearity
22
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Fig. 2. Full-scan product ion spectra of [M+H]+ for nuciferine (A) and papaverine (IS, B).
was obtained by plotting the peak area ratios of nuciferine to the IS versus the concentrations of nuciferine and assessed by weighted least-squares linear regression using 1/x2 as weighting factor. Accuracy and precision were evaluated in five replicates at three QC levels on the same day and three batches on three consecutive validation days. The intra- and inter-day precision (RSD) should not exceed 15% and the accuracy (RE) should be within ±15%. The extraction recovery was determined at three QC levels and calculated by comparing the standard peak areas of the analytes obtained from the extracted samples with post-extracted samples spiked with the analytes. The matrix effect was determined by comparing the standard peak areas of the analytes dissolved with the blank matrix extract against those dissolved with mobile phase at corresponding concentrations. Both the precision (RSD) of extraction recovery and matrix effect should be no more than 15%.
The stability was investigated by analyzing five replicates of stable samples at three QC levels under different conditions: 24 h storage in the autosamper, 12 h exposure at room temperature, three freeze/thaws cycles, and storage at −80 ◦ C for 30 days. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% R.E.) and precision (15% R.S.D). Carry-over was assessed following injection of a blank biosample immediately after three repeats of the upper limit of quantification (ULOQ) concentration (100 ng/ml), and the response of all carry-over peaks must be within 15% of the LLOQ response. The levels of some plasma samples exceeded the highest concentration of the calibration curve. The dilution integrity experiment must be carried out by a 2-fold dilution of ULOQ concentration (100 ng/ml × 2) with blank plasma for five replicates [22]. Accuracy within ±15% and precision less than 15% were considered acceptable.
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
2.6. Pharmacokinetic and tissue distribution study Sprague-Dawley male rats weighing 250–300 g were kept in an environmentally controlled breeding room maintained at a temperature of (22 ± 2) ◦ C and relative humidity of (50 ± 10) % for one
23
week prior to the start of the experiments, and fed with standard laboratory food and water. All rats were fasted overnight with access to water only before being dosed. Nuciferine was dissolved in normal saline to prepare 0.04 mg/ml solution for intravenous administration, while nuciferine was
Fig. 3. Representative MRM chromatograms of blank plasma (A), plasma spiked with nuciferine at LLQQ and IS (B), plasma sample (C), heart sample (D), liver sample (E), spleen sample (F), lung sample (G), kidney sample (H), brain sample (I) and adipose tissue sample (J) obtained 12 h after an intravenous administration of 0.2 mg/kg nuciferine.
24
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Fig. 3. (Continued ).
dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na, Sigma, USA) solution to prepare 0.4, 1.0, 2.0 mg/ml suspension for oral administration. Dosing solutions were prepared on the day of the experiment. For the pharmacokinetic study, 24 rats were randomly assigned to four groups (6 rats/group) for intravenous (IV) administration of 0.2 mg/kg nuciferine or oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine, respectively. Approximately 200 l blood was collected from the ocular vein of each rat into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h and 24 h after IV administration, and 0 (pro-drug), 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 6 h, 9 h, 12 h and 24 h after oral administration. The blood samples were immediately centrifuged at 8000 rpm for 5 min. The resulting plasma layers were separated and then stored at −80 ◦ C until analysis. The pharmacokinetic parameters of nuciferine were calculated by PKSolver V2.0 [23]. The bioavailability was calculated as follows: F = (AUCoral × Doseintravenous )/(AUCintravenous × Doseoral ) × 100%. For tissue distribution study, 24 rats were randomly assigned to four groups (6 rats/group) to carry out tissue distribution study. Rats in the four groups were sacrificed at 30 min, 2 h, 6 h and 12 h after IV injection of 0.2 mg/kg nuciferine. Subsequently, the heart,
liver, spleen, lung, kidney, brain and adipose tissue were immediately removed, washed in normal saline and blotted dry with filter paper. Meanwhile, blood samples were also collected from the carotid artery, and plasma was acquired according to the above method. An accurately weighed amount of the soft tissue sample (0.2 g) was individually homogenized with normal saline (0.8 ml) and stored at −80 ◦ C until analysis. 3. Results and discussion 3.1. Optimization of LC–MS/MS conditions and extraction method To optimize the ESI conditions for nuciferine and IS, quadrupole full scans were carried out in the positive or negative detection mode by infusing an approximately 100 ng/ml analyte in acetonitrile using a Harvard infusion pump (Harvard Apparatus, USA). ESI in both positive and negative ion modes was tested, and the results showed that ESI in the positive ions mode provided a higher sensitivity. Nuciferine and IS gave predominant singly charged protonated precursor [M+H]+ at m/z of 296.1 and 340.2 in Q1 full scan mode, respectively. The major fragment ions were observed in the product spectrum at m/z 265, 250 and 234 for nuciferine when
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
25
CE was 24 eV (Fig. 2). The m/z 265 fragment was dominant and therefore used for its quantification of nuciferine. Under similar procedures, the optimum MRM transition of IS was chosen as m/z 340.2 → 202.2 when CE was 36 eV. The mobile phase played an important role in achieving good chromatographic behaviors including peak symmetry and sensitivity. It was observed that acetonitrile gave a better peak shape and a lower background noise than methanol as the organic phase. Both nuciferine and IS were observed to have a higher response and a decrease in the tailed chromatographic peak when 5 mM ammonium acetate was added in the mobile phase. Finally, acetonitrile and water containing 5 mM ammonium acetate were used as the mobile phase in the isocratic model at a flow rate of 0.26 ml/min. The protein precipitation method was firstly considered for sample preparation in the present study. The matrix effect was observed in LC–MS/MS but the ratio did not exceed 80%, which could not meet the analytical requirement. Therefore, liquid-liquid extraction was chosen to prevent matrix effect. Different extracting solvents such as chloroform, ethyl acetate, ethylether, isopropanol and dichloromethane were investigated and ethyl acetate appeared to be the best choice, yielding a higher extraction ratio and lower background interference. It was also found that alkalinizing ethyl acetate with 5.3% Na2 CO3 solution could achieve a more satisfactory recovery effect for nuciferine and IS.
Table 1 Calibration curve, correlation coefficients and linear ranges of nuciferine in different bio-samples.
3.2. Method validation
3.2.3. Precision and accuracy The results for intra- and inter-day precision and accuracy are presented in Table 2. Throughout the three QC concentrations examined, the intra- and inter-day accuracy was measured to be within −9.5% and 9.9% respectively, while the intra- and inter-day precision was less than 11.8%, indicating that the precision and accuracy of this assay are within the acceptable range of analysis.
3.2.1. Selectivity No endogenous interference was observed in the plasma or tissue sample at retention time of nuciferine and IS because of the high selectivity of MRM mode. Representative chromatograms obtained from the blank rat plasma, blank rat plasma spiked with
Bio-sample
Calibration curve
Correlation coefficient (r)
Plasma Heart Liver Spleen Lung Kidney Brain Adipose
Y = 0.0788X + 0.0207 Y = 0.0917X + 0.0224 Y = 0.107X + 0.0562 Y = 0.107X + 0.0142 Y = 0.0945X + 0.0305 Y = 0.124X + 0.0269 Y = 0.12X + 0.0207 Y = 0.0424X + 0.0281
0.9969 0.9954 0.9966 0.9974 0.9962 0.9971 0.9951 0.9954
Linear range (ng/ml) 0.1–100 0.1–10 0.1–50 0.1–50 0.1–100 0.1–50 0.1–50 0.1–10
the nuciferine at LLOQ level (0.1 ng/ml) and IS, and plasma and tissue sample at 12 h after IV injection are shown in Fig. 3. 3.2.2. Calibration curves and lower limits of quantification The calibration curves, correlation coefficients and linear ranges of nuciferine in plasma and each tissue are listed in Table 1. The calibration curves had a correlation coefficient (r) of 0.995 or better. LLOQ was 0.1 ng/ml (S/N > 10) for all biosamples and 10 fold more sensitive than Xie’s method [18], which are sufficient for the pharmacokinetics and tissue distribution study of low dose nuciferine.
Table 2 Summary of the inter- and intra-day variability for the assay of nuciferine in rat plasma and tissues. Bio-sample
Nominal concentrations (ng/ml)
Inter-day (n = 5)
Intra-day (n = 15)
Measured concentrations (ng/ml)
Precision R.S.D (%)
Accuracy R.E. (%)
Measured concentrations (ng/ml)
Precision R.S.D (%)
Accuracy R.E. (%)
0.25 2.5 80
0.26 ± 0.02 2.30 ± 0.16 77.5 ± 2.98
6.4 6.9 3.8
2.2 −7.9 −3.1
0.25 ± 0.03 2.29 ± 0.18 77.3 ± 6.37
11.6 7.9 8.2
−1.2 −8.2 −3.4
Heart
0.25 1 8
0.26 ± 0.02 0.92 ± 0.09 8.68 ± 0.70
6.6 9.9 8.1
5.2 −8.0 8.6
0.27 ± 0.02 0.96 ± 0.11 8.79 ± 0.92
7.9 11.8 10.5
6.0 −4.3 9.9
Liver
0.25 2.5 40
0.23 ± 0.02 2.46 ± 0.24 42.4 ± 3.60
7.6 9.7 8.5
−8.3 −1.5 5.9
0.24 ± 0.03 2.43 ± 0.17 42.9 ± 2.23
8.0 7.1 5.2
−2.2 −2.7 7.3
Spleen
0.25 2.5 40
0.23 ± 0.02 2.37 ± 0.11 37.6 ± 2.74
7.6 4.6 7.3
−8.9 −5.1 −6.0
0.24 ± 0.03 2.38 ± 0.19 36.3 ± 3.45
10.8 7.8 9.5
−3.2 −5.0 −9.4
Lung
0.25 2.5 80
0.26 ± 0.02 2.36 ± 0.13 77.6 ± 6.19
8.7 5.5 8.0
4.8 −5.7 −2.9
0.27 ± 0.02 2.42 ± 0.23 77.5 ± 7.11
6.3 9.5 9.2
7.2 −3.4 −3.2
Kidney
0.25 2.5 40
0.26 ± 0.01 2.43 ± 0.14 38.8 ± 3.31
5.1 5.6 8.5
5.2 −2.7 −3.1
0.26 ± 0.02 2.56 ± 0.12 37.2 ± 3.58
7.7 4.9 9.6
4.7 2.3 −7.0
Brain
0.25 2.5 40
0.25 ± 0.02 2.58 ± 0.13 38.1 ± 2.95
9.2 5.2 7.7
1.9 3.2 −4.7
0.27 ± 0.02 2.66 ± 0.18 38.6 ± 3.68
8.6 6.7 9.5
8.3 6.4 −3.4
0.25 1 8
0.25 ± 0.01 0.90 ± 0.05 8.79 ± 0.54
5.7 5.8 6.2
0.9 −9.5 9.9
0.26 ± 0.02 0.91 ± 0.08 8.60 ± 0.73
9.4 9.3 8.5
5.5 −9.2 7.4
Plasma
Adipose
26
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Table 3 Extraction recovery and matrix effect of nuciferine and IS in rat plasma and tissue homogenates (n = 5). Bio-sample
Nominal concentrations (ng/ml)
RSD (%)
Matrix effect (%)
RSD (%)
Plasma
0.25 2.5 80
99.4 ± 9.4 90.4 ± 6.2 88.4 ± 4.8
9.4 6.9 5.4
107.8 ± 5.3 109.3 ± 6.5 110.2 ± 6.2
4.9 6.0 5.7
Heart
0.25 1 8
103.5 ± 6.8 97.5 ± 5.9 103.3 ± 6.9
6.6 6.1 6.7
98.9 ± 7.7 107.3 ± 4.8 109.8 ± 4.1
7.8 4.5 3.7
Liver
0.25 2.5 40
95.5 ± 7.8 104.9 ± 8.6 93.7 ± 7.8
8.2 8.2 8.3
107.6 ± 8.2 106.2 ± 9.3 109.6 ± 5.3
7.6 8.8 4.9
Spleen
0.25 2.5 40
90.4 ± 5.3 99.8 ± 8.8 91.1 ± 5.4
5.9 8.8 5.9
99.6 ± 3.8 101.1 ± 8.1 98.3 ± 7.1
3.8 8.0 7.2
Lung
0.25 2.5 80
100.8 ± 4.5 86.9 ± 3.3 90.3 ± 6.8
4.4 3.8 7.6
102.7 ± 5.8 105.0 ± 9.4 106.8 ± 7.9
5.6 9.0 7.4
Kidney
0.25 2.5 40
97.6 ± 7.5 87.3 ± 5.6 100.9 ± 10.0
7.7 6.4 9.9
105.1 ± 9.6 100.0 ± 5.5 102.5 ± 9.1
9.2 5.5 8.9
Brain
0.25 2.5 40
101.6 ± 5.9 94.4 ± 6.1 102.7 ± 8.5
5.8 6.5 8.3
98.9 ± 9.4 111.3 ± 5.8 104.0 ± 6.7
9.5 5.2 6.5
0.25 1 8
98.7 ± 7.3 92.4 ± 5.1 100.8 ± 6.4
7.4 5.5 6.4
104.1 ± 9.1 101.8 ± 6.0 103.6 ± 7.4
8.7 5.9 7.1
Adipose
Extraction recovery (%)
3.2.4. Extraction recovery and matrix effect The extraction recovery and matrix effect determined for nuciferine are shown in Table 3. All extraction recoveries of nuciferine at three QC concentrations as well as IS at 120 ng/ml in biosamples were more than 85%, which could meet the requirements of analysis. The absolute matrix effect of nuciferine ranged from 98.3% to 111.3%, and the absolute matrix effect of IS was less than 115% in the biological matrixes, indicating that there was no obvious matrix effect for nuciferine and IS in the plasma or tissue sample for this LC–MS/MS determination.
profile of nuciferine for both IV and oral administration [24]. The corresponding pharmacokinetic parameters are listed in Table 5. Upon IV administration at the dose of 0.2 mg/kg, nuciferine was shown to have a moderate apparent volume of distribution (Vd = 1.5 ± 0.5 L/kg), a short half-life time (t1/2 = 0.6 ± 0.2 h) and a clearance of (1.8 ± 0.5) L/h/kg. After three dose levels of oral
3.2.5. Stability The stability test was carried out under various conditions that the samples may experience. The results shown in Table 4 demonstrate that nuciferine was stable in the rat plasma and tissue homogenate after three freeze–thaw cycles, at room temperature for 12 h, at 4 ◦ C in the auto-sampler for 24 h, and in a long term freezer set at −80 ◦ C for 30 days. It is noteworthy that no significant degradation of nuciferine was observed under the above conditions. 3.2.6. Carry-over and dilution No detectable carry-over was observed in the current LC–MS/MS method. The results of the dilution integrity experiments indicate that the accuracy was within ±13.0% and the precision was less than 6.9%, which could meet the requirements of dilution analysis. 3.3. Pharmacokinetic study The validated method for the quantitation of nuciferine in rat plasma was developed with a low LLOQ of 0.1 ng/ml and applied to a pharmacokinetic study in rats after oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine and IV administration of 0.2 mg/kg nuciferine. The mean plasma concentration-time curves are shown in Fig. 4. The one-compartment model provided the best fit due to its lower AIC than the two-compartment model, indicating that the former model is more suitable to describe the pharmacokinetic
Fig. 4. Mean plasma concentration-time curve of nuciferine after oral administration (A) of 2 mg/kg, 5 mg/kg, 10 mg/kg nuciferine or intravenous administration (B) of 0.2 mg/kg nuciferine in rats. (n = 6, mean ± SD).
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
27
Table 4 Stability of nuciferine in rat plasma and tissues sample (n = 5). Autosampler (24 h, 4 ◦ C)
Three freeze/thaw cycles
Room temperature (12 h)
Long term (30 days, −80 ◦ C)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
−5.8 −3.1 4.7
10.0 7.5 9.6
1.2 −3.3 3.7
9.6 8.0 8.1
−8.2 −3.0 6.3
10.9 8.3 10.5
−7.5 −4.3 −1.9
6.0 −2.1 −1.8
11.7 10.0 12.6
2.1 −2.2 −4.5
9.9 7.3 7.3
2.6 −5.4 −5.8
10.9 6.4 12.1
−0.5 −2.7 −3.1
6.8 6.3 5.7
2.8 −2.2 −9.0
8.5 8.2 6.7
1.2 1.4 −7.5
9.1 9.2 9.7
−4.1 3.2 −2.2
7.7 7.3 10.8
−1.6 −2.9 −4.5
0.25 2.5 40
6.7 5.2 10.2
8.4 −5.3 3.4
4.9 12.1 6.9
5.6 −0.5 −4.4
9.4 5.4 9.4
−4.2 1.3 −1.6
6.2 6.3 9.1
−2.5 −3.2 −6.8
Lung
0.25 2.5 80
9.9 7.4 8.2
−3.3 −0.5 −1.7
8.9 5.9 5.3
−5.1 −3.7 −3.5
6.5 6.1 2.9
1.4 0.5 −1.0
4.6 7.2 3.0
−2.0 −2.9 −0.2
Kidney
0.25 2.5 40
9.9 8.1 10.6
−2.8 9.1 9.1
6.8 8.6 9.4
−2.2 1.4 7.2
9.5 7.9 9.3
0.6 4.8 6.0
5.7 7.1 8.0
−2.2 1.1 −3.2
Brain
0.25 2.5 40
10.1 4.5 7.6
0.6 2.8 2.5
5.4 8.0 12.4
6.0 3.3 4.4
9.2 11.4 12.9
5.1 1.3 2.7
5.1 7.6 7.2
−3.6 −3.0 0.9
0.25 1 8
9.0 11.7 8.0
−4.9 3.2 10.0
7.1 9.7 9.3
−4.0 2.5 8.0
7.3 5.1 11.5
−4.5 1.1 8.4
6.1 11.1 11.5
−3.5 3.4 3.1
Biological fluids
Nominal concentration (ng/ml)
Plasma
0.25 2.5 80
5.6 8.0 9.0
Heart
0.25 1 8
6.4 8.8 8.6
Liver
0.25 2.5 40
Spleen
Adipose
administration, Cmax , AUC0–24 h and AUC0–∞ versus the nuciferine dose profiles were linearly with a correlation coefficients of 0.979, 0.985 and 0.988 respectively, showing dose-dependent pharmacokinetics. There was no statistically significant difference in Tmax , t1/2 , Cl and Vd (two tailed paired test, p > 0.05). The value of Tmax and t1/2 indicated that nuciferine was rapidly distributed. The apparent volume of distribution (Vd ) implied that nuciferine exhibited a obvious tissue uptake after oral administration. The values of absolute bioavailability of nuciferine were (3.8 ± 1.4)%, (4.2 ± 1.3)% and (3.9 ± 1.0)% for oral doses of 2.0, 5.0 and 10.0 mg/kg, respectively. These results suggested that its bioavailability was independent of the doses. It was also concluded that nuciferine might have a low absolute bioavailability. Our previous study on absorption profiles of nuciferine in rat intestine [25] showed that the absorption rate constant in the duodenum, jejunum, ileum and colon was 0.014, 0.020, 0.022 and 0.019 min−1 , respectively, indicating that nuciferine had bad absorption from the gastrointestinal tract in rats. Thus, the low oral
bioavailability can be rationalized. Thus, nanoparticle drug delivery system was further investigated to enhance pharmacological activities of nuciferine after oral administration.
3.4. Tissue distribution study Nuciferine distributions in the heart, liver, spleen, lung, kidney, brain and adipose tissue are listed in Fig. 5. The highest concentration was observed at 0.5 h in the lung, followed by the kidney, spleen, brain, liver, adipose tissue and heart after a single dose of IV administration of 0.2 mg/kg nuciferine. Compared with the concentration in plasma, nuciferine could be detected in various tissue samples, indicating that it had a relatively high degree of tissue distribution, which is consistent with the result of the pharmacokinetic study. The sample collected 12 h after administration showed that nuciferine was cleared gradually and no accumulation was observed in the tissues. The higher concentration in the lung,
Table 5 One-compartmental pharmacokinetic parameters of nuciferine after oral administration of 2, 5, 10 mg/kg nuciferine or intravenous administration of 0.2 mg/kg nuciferine in rats (n = 6, mean ± SD). Pharmacokinetic parameters
Unit
Value Oral administration
2 mg/kg t1/2 Vd Cl Tmax Cmax AUC0–24 h AUC0–∞ Bioavailability
h L/kg L/h/kg h ng/ml ng/ml × h ng/ml × h %
1.5 112.4 55.5 0.9 13.5 41.7 41.7 3.8
± ± ± ± ± ± ± ±
Intravenous administration 5 mg/kg
0.8 57.9 22.0 0.4 5.1 19.1 19.1 1.4
0.8 57.3 58.6 0.6 63.4 118.2 118.2 4.2
± ± ± ± ± ± ± ±
10 mg/kg 0.4 24.3 39.9 0.2 19.0 52.5 52.5 1.3
1.0 64.1 52.5 0.5 103.0 207.1 207.5 3.9
± ± ± ± ± ± ± ±
0.6 27.1 15.6 0.1 15.7 67.5 68.3 1.0
0.2 mg/kg 0.6 ± 1.5 ± 1.8 ± – – 119.0 ± 119.0 ± –
0.2 0.5 0.5
31.4 31.4
28
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
reformation and the hypothalamus pathway-mediated antiobesity effect of nuciferine. Acknowledgements This study was supported by the National Science and Technology Major Projects of the Twelfth Five-year Plan (No. 2011ZX09302-007-02), Research Fund for the Doctoral Program of Higher Education of China (No. 20110073120097), and the Research Fund of Shanghai JiaoTong University School of Medicine (No. JYY0906). References
Fig. 5. Mean concentration of nuciferine in various tissues at 30 min, 2 h, 6 h and 12 h after an intravenous administration of 0.2 mg/kg nuciferine (n = 6, mean ± SD).
kidney and spleen might be attributed to the high blood flow in these organs. A previous study [13] showed that 80% cholesterol ester associated with lipoproteins was produced by the liver, and nuciferine could clearly suppress HFD-induced alterations in the expression of genes involved in lipid metabolism in the liver to treat dyslipidemia. Siegner et al. [5] also found that adipocytes incubated with the lotus leaf extract with the major constituent of nuciferine exhibited a significant increase in lipolysis-activity and a decrease in adipocyte differentiation capacity, confirming that the liver and adipose tissue were the targets of nuciferine to exert the antiobesity and hypolipidemic effects. The result showed that nuciferine was distributed into the liver and adipose tissue, which provides the material basis for its pharmacological actions. The concentration of nuciferine in the brain tissue was almost equal to that in plasma, suggesting that nuciferine could cross the blood–brain barrier. This finding was potentially important, knowing that the hypothalamus controls the appetite through the feeding center of the brain [26]. As the hypothalamus may be another potential target for the anti-obesity effect of nuciferine, it could be a new field of future research. 4. Conclusion A LC–MS/MS method for the quantification of nuciferine in rat plasma and tissue samples has been established and successfully used to evaluate the pharmacokinetic profile and tissue distribution after oral and IV administration. The method showed high sensitivity, specificity and reliability with an LLOQ of 0.1 ng/ml. The results obtained from the pharmacokinetic and tissue distribution study suggest that nuciferine distribution in the brain, liver and adipose tissue could conduce to the therapeutic effects, but the low absolute bioavailability may limit its further application as an anti-obesity agent. Our ongoing study would focus on the pharmaceutical
[1] Editorial Committee of the Pharmacopoeia of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, Beijing, China, 2010. [2] P.K. Mukherjee, D. Mukherjee, A.K. Maji, S. Rai, M. Heinrich, J. Pharm. Pharmacol. 61 (2009) 407–422. [3] H. Du, J.S. You, X. Zhao, J.Y. Park, S.H. Kim, K.J. Chang, J. Biomed. Sci. 17 (2010) S42. [4] Y. Ono, E. Hattori, Y. Fukaya, S. Imai, Y. Ohizumi, J. Ethnopharmacol. 106 (2006) 238–244. [5] R. Siegner, S. Heuser, U. Holtzmann, J. Söhle, A. Schepky, T. Raschke, et al., Nutr. Metab. 7 (2010) 66. [6] X.B. Luo, B. Chen, J.J. Liu, S.Z. Yao, Anal. Chim. Acta 538 (2005) 129–133. [7] S. Nakamura, S. Nakashima, G. Tanabe, Y. Oda, N. Yokota, K. Fujimoto, T. Matsumoto, R. Sakuma, T. Ohta, K. Ogawa, S. Nishida, H. Miki, H. Matsuda, O. Muraoka, M. Yoshikawa, Bioorg. Med. Chem. 21 (2013) 779–787. [8] Y. Kashiwada, A. Aoshima, Y. Ikeshiro, Y.P. Chen, H. Furukawa, M. Itoigawa, T. Fujioka, K. Mihashi, L.M. Cosentino, S.L. Morris-Natschke, K.H. Lee, Bioorg. Med. Chem. 13 (2005) 443–448. [9] J. Boustie, J.L. Stigliani, J. Montanha, M. Amoros, M. Payard, L. Girre, J. Nat. Prod. 61 (1998) 480–484. [10] Z.D. Yang, X. Zhang, J. Du, Z.J. Ma, F. Guo, S. Li, X.J. Yao, Nat. Prod. Res. 26 (2012) 387–392. [11] K.H. Nguyen, T.N. Ta, T.H. Pham, Q.T. Nguyen, H.D. Pham, S. Mishra, B.L. Nyomba, J. Ethnopharmacol. 142 (2012) 488–495. [12] X. Han, W. Wang, C. Wu, S. Liu, L. Wang, Progr. Modern Biomed. 8 (2008) 1628–1630. [13] F. Guo, X. Yang, X. Li, R. Feng, C. Guan, Y. Wang, Y. Li, C. Sun, PLoS ONE 8 (2013) e63770. [14] X.O. Wu, Y. Xiong, China Pharm. 7 (2004) 262–264. [15] Y.X. Wang, B. Liu, R.B. Shi, Zhongguo Zhong Yao Za Zhi 33 (2008) 1713–1716. [16] D.F. Ling, S.L. Peng, J. Anhui Agric. Sci. 36 (2008) 10301–10302. [17] T.C. Do, T.D. Nguyen, H. Tran, H. Stuppner, M. Ganzera, J. Chromatogr. A 1302 (2013) 174–180. [18] S.L. Xie, Y.Y. Wang, Y.C. Tan, P.F. Lu, C.J. Tian, R.K. Dai, J. Guangdong Coll. Pharm. 1 (2010) 17–19. [19] M.X. Lin, G.R. Lin, J.F. Chen, Y.H. Guo, J. Fuzhou Univ. 36 (2008) 303–307. [20] Food and Drug Administration of the United States, Guidance Industry Bioanalytical Method Validation, United States. for http://www.fad.gov/cder/guidance/index.hem (accessed May 2001). [21] H. Zhu, X. Zhang, J. Guan, B. Cui, L. Zhao, X. Zhao, J. Pharm. Biomed. 78–79 (2013) 136–140. [22] B. Ma, X. Li, J. Li, Q. Zhang, Y. Liu, X. Yang, et al., J. Pharm. Biomed. 88 (2014) 191–200. [23] Y. Zhang, M. Huo, J. Zhou, S. Xie, P.K. Solver, Comput. Methods Progr. Biomed. 99 (2010) 306–314. [24] K. Balakumar, C.V. Raghavan, N.T. Selvan, R.H. Prasad, S. Abdu, Colloids Surf. B: Biointerf. 112 (2013) 337–343. [25] X.Y. Li, E.J. Zhou, S.Y. Gu, Y. Xu, G.L. Liu, Chin. Pharm. J. J46 (2011) 1417–1420. [26] M.S. Byerly, R.D. Swanson, N.N. Semsarzadeh, P.S. McCulloh, K. Kwon, S. Aja, T.H. Moran, G.W. Wong, S. Blackshaw, Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (2013) R1085–R1095.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
A sensitive liquid chromatography–tandem mass spectrometry method for pharmacokinetics and tissue distribution of nuciferine in rats Shengying Gu, Guanhua Zhu, Yuzhu Wang, Qin Li, Xin Wu, Jigang Zhang, Gaolin Liu ∗ , Xiaoyu Li ∗ Department of Clinic Pharmacy, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 24 February 2014 Accepted 22 April 2014 Available online 5 May 2014 Keywords: Nuciferine LC–MS/MS Pharmacokinetics Tissue distribution
a b s t r a c t Nuciferine is an important drug candidate for the treatment of obesity-related diseases. However, few investigations have been conducted about the pharmacokinetics and tissue distribution of nuciferine to better understand its behavior and action mechanism in vivo. Thus, a sensitive and reliable liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) method was established and validated for the quantification of nuciferine in rat plasma and tissue samples. The validated method was successfully applied to the pharmacokinetic and tissue distribution study of nuciferine in rats. One-compartmental pharmacokinetic parameters indicated that nuciferine had rapid distribution, extensive tissue uptake, and poor absorption into systemic circulation. The values of absolute bioavailability were (3.8 ± 1.4)%, (4.2 ± 1.3)% and (3.9 ± 1.0)% after oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine and intravenous administration of 0.2 mg/kg nuciferine in rats. The results of the tissue distribution study suggested that nuciferine was distributed into the brain, liver and adipose tissue after intravenous administration. In conclusion, the present study may provide a material basis for study of the pharmacological action of nuciferine in the treatment of obesity, and meaningful insights into further study on dosage modification. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lotus (Nelumbo nucifera Gaertn) leaf is a traditional Chinese medicine used for clearing heat, removing heatstroke, cooling and stanching blood flow [1,2]. Recent pharmacological studies have demonstrated that lotus leaf also has an anti-obesity effect by inhibiting the absorption of lipids and carbohydrates, accelerating lipid metabolism [3,4], and reducing triglyceride accumulation in adipocytes [5]. This effect is supposed to be attributed to alkaloid nuciferine, the major constituent extracted from lotus leaf [6]. Nuciferine ((R)-1, 2-dimethoxyaporphine) is an aromatic ethercontaining compound whose chemical structure is shown in Fig. 1. Previous studies reported that nuciferine possessed anti-melanogenesis [7], anti-HIV [8], anti-poliovirus [9], antiacetylcholinesterase [10] and insulin secretagogue activities [11]. Recent studies have demonstrated that nuciferine can ameliorate
∗ Corresponding authors at: 650# New Songjiang Road, Songjiang District, Shanghai 200080, People’s Republic of China. Tel.: +86 21 37798312. E-mail addresses: [email protected] (G. Liu), [email protected] (X. Li). http://dx.doi.org/10.1016/j.jchromb.2014.04.038 1570-0232/© 2014 Elsevier B.V. All rights reserved.
hyperlipemia, lower cholesterols and improve hepatic lipid metabolism [12,13]. Given these remarkable functions, more attention has been paid to the pharmacological research on nuciferine and its clinical therapeutic application. Pharmacokinetics and characteristics of tissue distribution are vital to the understanding of the in vivo behavior and action mechanism. So there is an increasing demand on quality control of nuciferine. Several techniques have been employed for analysis of nuciferine, such as thin-layer chromatography (TLC) [14], high-performance liquid chromatography (HPLC) with ultraviolet (UV) [15], HPLC coupled with mass spectrometry (MS) [16] and non-aqueous capillary electrophoresis using UV/MS [17]. However, most of these methods are time-consuming and lack complete validation data or sensitive pharmacokinetics study. Xie et al. [18] developed a HPLC–MS/MS method with the lower limit of quantification (LLOQ) of 1 ng/ml and applied it to the pharmacokinetics study of nuciferine after oral administration of 20 mg/kg in rats. However, the pharmacodynamic study by Lin et al. [19] reported that the daily intake of nuciferine was (3.2–12.8) mg/kg in rats, suggesting that the pharmacokinetics study of Xie et al. might be of little guiding significance and the sensitivity of Xie’s HPLC–MS/MS
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
21
Fig. 1. The chemical structure of nuciferine and papaverine (IS).
method could not meet the requirement of low-dose nuciferine. The goal of this study was to develop and validate a sensitive and reliable reversed phase LC–MS/MS method and applied it to study pharmacokinetics and tissue distribution of nuciferine in rats, hoping that it could provide helpful information for the development of suitable dosage forms and clinical application of nuciferine. 2. Experimental 2.1. Chemicals and materials Nuciferine (purity ≥ 98.0%) was purchased from ZR Chemicals (Shanghai, China). Papaverine as internal standard (IS, purity ≥ 99.9%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). LC–MS grade ammonium acetate was purchased from CNW Technologies GmbH (Dusseldorf, Germany). HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). HPLC grade ethylacetate and sodium carbonate anhydrous were purchased from Aladdin (China). Water was purified by employing a Milli-Q water purification system (Millipore, France). 2.2. Instrumentation and LC–MS/MS method The LC–MS/MS system consisted of a Shimadzu LC-20A chromatographic system (Shimadzu, Japan) and an API 4000 mass spectrometer (AB SCIEX, Singapore) equipped with electrospray ionization (ESI) source system. The system control and data analysis were performed by AB Sciex Analyst software (the software version: Analyst 1.5.1). Chromatographic separation was carried out on a Shim-pack XD-ODS column (50 mm × 3 mm, 2.2 m) with a Shim-pack GVDODS (5.0 mm × 2.0 mm, 2.2 m) guard column (Shimadzu, Japan). The HPLC was operated with a gradient mobile phase system consisting of water containing 5 mM ammonium acetate (phase A) and acetonitrile (phase B) at a flow rate of 0.26 ml/min. The pump was programmed as follows: phase B was increased from 15% to 60% within the first 3.5 min, held for 1 min, decreased to 15% within the next 1.5 min, and held for 0.5 min (total gradient time: 6.5 min). A 5 l sample was injected into the system with the auto-sampler conditioned at 4 ◦ C and column temperature maintained at 35 ◦ C. The mass spectrometer was operated in a positive ion mode. The multiple reaction monitoring (MRM) transitions were performed at m/z 296.1 → 265.0 for nuciferine and m/z 340.2 → 202.2 for papaverine (IS). Optimized values for declustering potential (DP), collision energy (CE), entrance potential (EP) and collision exit potential (CXP) were 80 V, 24 eV, 10 V and 15 V for nuciferine, and 90 V, 36 eV, 10 V and 15 V for IS, respectively. The MS parameters
were as follows: curtain gas of 12 psi; Ion spray voltage of 5500 V; source temperature of 500 ◦ C; Ion source Gas 1 and Gas 2 (nitrogen) of 40 psi and 50 psi. 2.3. Standard solutions, calibration and quality control samples Stock solutions were prepared by dissolving nuciferine and IS in methanol to obtain the concentrations of 1.0 and 1.2 mg/ml, respectively. Subsequently, a series of standard working solutions was prepared by diluting stock solution with methanol to get the concentrations of 0.1–100 ng/ml. A 120 ng/ml solution for IS was prepared in methanol. Calibration curves were prepared by spiking with 10 l different concentrations of the standard working stock solutions, 10 l IS solution (papaverine 120 ng/ml) and 100 l blank rat plasma or tissue homogenate sample to 1.5 ml Eppendorf tube on the analysis day. Calibration curves were prepared at concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 ng/ml for the plasma and lung; 0.1, 0.25, 0.5, 1, 2.5, 5 and 10 ng/ml for the heart and adipose tissue; 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 ng/ml for the liver, spleen, kidney and brain, respectively. The similar method was also used to prepare quality control samples (low QC, med QC and high QC) in rat plasma or tissue homogenates at concentrations of 0.25, 5 and 80 ng/ml for the plasma and lung; 0.25, 1 and 8 ng/ml for the heart and adipose tissue; 0.25, 2.5 and 40 ng/ml for the liver, spleen, kidney and brain. 2.4. Sample preparation A rat plasma or tissue homogenate sample (100 l) was placed in a 1.5 ml Eppendorf tube and mixed with 30 l 5.3% Na2 CO3 solution and 10 l IS solution (papaverine 120 ng/ml) for 3 min by vortexing. The mixture was extracted with ethyl acetate (1 ml) by vortexing and then centrifuged at 13,000 rpm for 5 min. The supernatant (900 l) was transferred to a new 1.5 ml Eppendorf tube and evaporated to dryness under vacuum. The dried residue was reconstituted with 100 l acetonitrile, vortex-mixed for 3 min and centrifuged at 13,000 rpm for 5 min. Finally, the supernatant liquid (5 l) was injected into the LC–ESI-MS/MS system. 2.5. Method validation This HPLC–MS/MS method was validated in accordance with the guidelines for Bioanalytical Method Validation published by the US FDA and other related guidelines with respect to specificity, linearity, precision and accuracy, recovery and matrix effect, stability, dilution and carry over [20,21]. Six blank rat plasma samples and tissue homogenate as a representative sample were used to evaluate the specificity. The linearity
22
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Fig. 2. Full-scan product ion spectra of [M+H]+ for nuciferine (A) and papaverine (IS, B).
was obtained by plotting the peak area ratios of nuciferine to the IS versus the concentrations of nuciferine and assessed by weighted least-squares linear regression using 1/x2 as weighting factor. Accuracy and precision were evaluated in five replicates at three QC levels on the same day and three batches on three consecutive validation days. The intra- and inter-day precision (RSD) should not exceed 15% and the accuracy (RE) should be within ±15%. The extraction recovery was determined at three QC levels and calculated by comparing the standard peak areas of the analytes obtained from the extracted samples with post-extracted samples spiked with the analytes. The matrix effect was determined by comparing the standard peak areas of the analytes dissolved with the blank matrix extract against those dissolved with mobile phase at corresponding concentrations. Both the precision (RSD) of extraction recovery and matrix effect should be no more than 15%.
The stability was investigated by analyzing five replicates of stable samples at three QC levels under different conditions: 24 h storage in the autosamper, 12 h exposure at room temperature, three freeze/thaws cycles, and storage at −80 ◦ C for 30 days. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% R.E.) and precision (15% R.S.D). Carry-over was assessed following injection of a blank biosample immediately after three repeats of the upper limit of quantification (ULOQ) concentration (100 ng/ml), and the response of all carry-over peaks must be within 15% of the LLOQ response. The levels of some plasma samples exceeded the highest concentration of the calibration curve. The dilution integrity experiment must be carried out by a 2-fold dilution of ULOQ concentration (100 ng/ml × 2) with blank plasma for five replicates [22]. Accuracy within ±15% and precision less than 15% were considered acceptable.
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
2.6. Pharmacokinetic and tissue distribution study Sprague-Dawley male rats weighing 250–300 g were kept in an environmentally controlled breeding room maintained at a temperature of (22 ± 2) ◦ C and relative humidity of (50 ± 10) % for one
23
week prior to the start of the experiments, and fed with standard laboratory food and water. All rats were fasted overnight with access to water only before being dosed. Nuciferine was dissolved in normal saline to prepare 0.04 mg/ml solution for intravenous administration, while nuciferine was
Fig. 3. Representative MRM chromatograms of blank plasma (A), plasma spiked with nuciferine at LLQQ and IS (B), plasma sample (C), heart sample (D), liver sample (E), spleen sample (F), lung sample (G), kidney sample (H), brain sample (I) and adipose tissue sample (J) obtained 12 h after an intravenous administration of 0.2 mg/kg nuciferine.
24
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Fig. 3. (Continued ).
dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na, Sigma, USA) solution to prepare 0.4, 1.0, 2.0 mg/ml suspension for oral administration. Dosing solutions were prepared on the day of the experiment. For the pharmacokinetic study, 24 rats were randomly assigned to four groups (6 rats/group) for intravenous (IV) administration of 0.2 mg/kg nuciferine or oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine, respectively. Approximately 200 l blood was collected from the ocular vein of each rat into heparinized tubes at 0 (pro-drug), 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h and 24 h after IV administration, and 0 (pro-drug), 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 6 h, 9 h, 12 h and 24 h after oral administration. The blood samples were immediately centrifuged at 8000 rpm for 5 min. The resulting plasma layers were separated and then stored at −80 ◦ C until analysis. The pharmacokinetic parameters of nuciferine were calculated by PKSolver V2.0 [23]. The bioavailability was calculated as follows: F = (AUCoral × Doseintravenous )/(AUCintravenous × Doseoral ) × 100%. For tissue distribution study, 24 rats were randomly assigned to four groups (6 rats/group) to carry out tissue distribution study. Rats in the four groups were sacrificed at 30 min, 2 h, 6 h and 12 h after IV injection of 0.2 mg/kg nuciferine. Subsequently, the heart,
liver, spleen, lung, kidney, brain and adipose tissue were immediately removed, washed in normal saline and blotted dry with filter paper. Meanwhile, blood samples were also collected from the carotid artery, and plasma was acquired according to the above method. An accurately weighed amount of the soft tissue sample (0.2 g) was individually homogenized with normal saline (0.8 ml) and stored at −80 ◦ C until analysis. 3. Results and discussion 3.1. Optimization of LC–MS/MS conditions and extraction method To optimize the ESI conditions for nuciferine and IS, quadrupole full scans were carried out in the positive or negative detection mode by infusing an approximately 100 ng/ml analyte in acetonitrile using a Harvard infusion pump (Harvard Apparatus, USA). ESI in both positive and negative ion modes was tested, and the results showed that ESI in the positive ions mode provided a higher sensitivity. Nuciferine and IS gave predominant singly charged protonated precursor [M+H]+ at m/z of 296.1 and 340.2 in Q1 full scan mode, respectively. The major fragment ions were observed in the product spectrum at m/z 265, 250 and 234 for nuciferine when
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
25
CE was 24 eV (Fig. 2). The m/z 265 fragment was dominant and therefore used for its quantification of nuciferine. Under similar procedures, the optimum MRM transition of IS was chosen as m/z 340.2 → 202.2 when CE was 36 eV. The mobile phase played an important role in achieving good chromatographic behaviors including peak symmetry and sensitivity. It was observed that acetonitrile gave a better peak shape and a lower background noise than methanol as the organic phase. Both nuciferine and IS were observed to have a higher response and a decrease in the tailed chromatographic peak when 5 mM ammonium acetate was added in the mobile phase. Finally, acetonitrile and water containing 5 mM ammonium acetate were used as the mobile phase in the isocratic model at a flow rate of 0.26 ml/min. The protein precipitation method was firstly considered for sample preparation in the present study. The matrix effect was observed in LC–MS/MS but the ratio did not exceed 80%, which could not meet the analytical requirement. Therefore, liquid-liquid extraction was chosen to prevent matrix effect. Different extracting solvents such as chloroform, ethyl acetate, ethylether, isopropanol and dichloromethane were investigated and ethyl acetate appeared to be the best choice, yielding a higher extraction ratio and lower background interference. It was also found that alkalinizing ethyl acetate with 5.3% Na2 CO3 solution could achieve a more satisfactory recovery effect for nuciferine and IS.
Table 1 Calibration curve, correlation coefficients and linear ranges of nuciferine in different bio-samples.
3.2. Method validation
3.2.3. Precision and accuracy The results for intra- and inter-day precision and accuracy are presented in Table 2. Throughout the three QC concentrations examined, the intra- and inter-day accuracy was measured to be within −9.5% and 9.9% respectively, while the intra- and inter-day precision was less than 11.8%, indicating that the precision and accuracy of this assay are within the acceptable range of analysis.
3.2.1. Selectivity No endogenous interference was observed in the plasma or tissue sample at retention time of nuciferine and IS because of the high selectivity of MRM mode. Representative chromatograms obtained from the blank rat plasma, blank rat plasma spiked with
Bio-sample
Calibration curve
Correlation coefficient (r)
Plasma Heart Liver Spleen Lung Kidney Brain Adipose
Y = 0.0788X + 0.0207 Y = 0.0917X + 0.0224 Y = 0.107X + 0.0562 Y = 0.107X + 0.0142 Y = 0.0945X + 0.0305 Y = 0.124X + 0.0269 Y = 0.12X + 0.0207 Y = 0.0424X + 0.0281
0.9969 0.9954 0.9966 0.9974 0.9962 0.9971 0.9951 0.9954
Linear range (ng/ml) 0.1–100 0.1–10 0.1–50 0.1–50 0.1–100 0.1–50 0.1–50 0.1–10
the nuciferine at LLOQ level (0.1 ng/ml) and IS, and plasma and tissue sample at 12 h after IV injection are shown in Fig. 3. 3.2.2. Calibration curves and lower limits of quantification The calibration curves, correlation coefficients and linear ranges of nuciferine in plasma and each tissue are listed in Table 1. The calibration curves had a correlation coefficient (r) of 0.995 or better. LLOQ was 0.1 ng/ml (S/N > 10) for all biosamples and 10 fold more sensitive than Xie’s method [18], which are sufficient for the pharmacokinetics and tissue distribution study of low dose nuciferine.
Table 2 Summary of the inter- and intra-day variability for the assay of nuciferine in rat plasma and tissues. Bio-sample
Nominal concentrations (ng/ml)
Inter-day (n = 5)
Intra-day (n = 15)
Measured concentrations (ng/ml)
Precision R.S.D (%)
Accuracy R.E. (%)
Measured concentrations (ng/ml)
Precision R.S.D (%)
Accuracy R.E. (%)
0.25 2.5 80
0.26 ± 0.02 2.30 ± 0.16 77.5 ± 2.98
6.4 6.9 3.8
2.2 −7.9 −3.1
0.25 ± 0.03 2.29 ± 0.18 77.3 ± 6.37
11.6 7.9 8.2
−1.2 −8.2 −3.4
Heart
0.25 1 8
0.26 ± 0.02 0.92 ± 0.09 8.68 ± 0.70
6.6 9.9 8.1
5.2 −8.0 8.6
0.27 ± 0.02 0.96 ± 0.11 8.79 ± 0.92
7.9 11.8 10.5
6.0 −4.3 9.9
Liver
0.25 2.5 40
0.23 ± 0.02 2.46 ± 0.24 42.4 ± 3.60
7.6 9.7 8.5
−8.3 −1.5 5.9
0.24 ± 0.03 2.43 ± 0.17 42.9 ± 2.23
8.0 7.1 5.2
−2.2 −2.7 7.3
Spleen
0.25 2.5 40
0.23 ± 0.02 2.37 ± 0.11 37.6 ± 2.74
7.6 4.6 7.3
−8.9 −5.1 −6.0
0.24 ± 0.03 2.38 ± 0.19 36.3 ± 3.45
10.8 7.8 9.5
−3.2 −5.0 −9.4
Lung
0.25 2.5 80
0.26 ± 0.02 2.36 ± 0.13 77.6 ± 6.19
8.7 5.5 8.0
4.8 −5.7 −2.9
0.27 ± 0.02 2.42 ± 0.23 77.5 ± 7.11
6.3 9.5 9.2
7.2 −3.4 −3.2
Kidney
0.25 2.5 40
0.26 ± 0.01 2.43 ± 0.14 38.8 ± 3.31
5.1 5.6 8.5
5.2 −2.7 −3.1
0.26 ± 0.02 2.56 ± 0.12 37.2 ± 3.58
7.7 4.9 9.6
4.7 2.3 −7.0
Brain
0.25 2.5 40
0.25 ± 0.02 2.58 ± 0.13 38.1 ± 2.95
9.2 5.2 7.7
1.9 3.2 −4.7
0.27 ± 0.02 2.66 ± 0.18 38.6 ± 3.68
8.6 6.7 9.5
8.3 6.4 −3.4
0.25 1 8
0.25 ± 0.01 0.90 ± 0.05 8.79 ± 0.54
5.7 5.8 6.2
0.9 −9.5 9.9
0.26 ± 0.02 0.91 ± 0.08 8.60 ± 0.73
9.4 9.3 8.5
5.5 −9.2 7.4
Plasma
Adipose
26
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
Table 3 Extraction recovery and matrix effect of nuciferine and IS in rat plasma and tissue homogenates (n = 5). Bio-sample
Nominal concentrations (ng/ml)
RSD (%)
Matrix effect (%)
RSD (%)
Plasma
0.25 2.5 80
99.4 ± 9.4 90.4 ± 6.2 88.4 ± 4.8
9.4 6.9 5.4
107.8 ± 5.3 109.3 ± 6.5 110.2 ± 6.2
4.9 6.0 5.7
Heart
0.25 1 8
103.5 ± 6.8 97.5 ± 5.9 103.3 ± 6.9
6.6 6.1 6.7
98.9 ± 7.7 107.3 ± 4.8 109.8 ± 4.1
7.8 4.5 3.7
Liver
0.25 2.5 40
95.5 ± 7.8 104.9 ± 8.6 93.7 ± 7.8
8.2 8.2 8.3
107.6 ± 8.2 106.2 ± 9.3 109.6 ± 5.3
7.6 8.8 4.9
Spleen
0.25 2.5 40
90.4 ± 5.3 99.8 ± 8.8 91.1 ± 5.4
5.9 8.8 5.9
99.6 ± 3.8 101.1 ± 8.1 98.3 ± 7.1
3.8 8.0 7.2
Lung
0.25 2.5 80
100.8 ± 4.5 86.9 ± 3.3 90.3 ± 6.8
4.4 3.8 7.6
102.7 ± 5.8 105.0 ± 9.4 106.8 ± 7.9
5.6 9.0 7.4
Kidney
0.25 2.5 40
97.6 ± 7.5 87.3 ± 5.6 100.9 ± 10.0
7.7 6.4 9.9
105.1 ± 9.6 100.0 ± 5.5 102.5 ± 9.1
9.2 5.5 8.9
Brain
0.25 2.5 40
101.6 ± 5.9 94.4 ± 6.1 102.7 ± 8.5
5.8 6.5 8.3
98.9 ± 9.4 111.3 ± 5.8 104.0 ± 6.7
9.5 5.2 6.5
0.25 1 8
98.7 ± 7.3 92.4 ± 5.1 100.8 ± 6.4
7.4 5.5 6.4
104.1 ± 9.1 101.8 ± 6.0 103.6 ± 7.4
8.7 5.9 7.1
Adipose
Extraction recovery (%)
3.2.4. Extraction recovery and matrix effect The extraction recovery and matrix effect determined for nuciferine are shown in Table 3. All extraction recoveries of nuciferine at three QC concentrations as well as IS at 120 ng/ml in biosamples were more than 85%, which could meet the requirements of analysis. The absolute matrix effect of nuciferine ranged from 98.3% to 111.3%, and the absolute matrix effect of IS was less than 115% in the biological matrixes, indicating that there was no obvious matrix effect for nuciferine and IS in the plasma or tissue sample for this LC–MS/MS determination.
profile of nuciferine for both IV and oral administration [24]. The corresponding pharmacokinetic parameters are listed in Table 5. Upon IV administration at the dose of 0.2 mg/kg, nuciferine was shown to have a moderate apparent volume of distribution (Vd = 1.5 ± 0.5 L/kg), a short half-life time (t1/2 = 0.6 ± 0.2 h) and a clearance of (1.8 ± 0.5) L/h/kg. After three dose levels of oral
3.2.5. Stability The stability test was carried out under various conditions that the samples may experience. The results shown in Table 4 demonstrate that nuciferine was stable in the rat plasma and tissue homogenate after three freeze–thaw cycles, at room temperature for 12 h, at 4 ◦ C in the auto-sampler for 24 h, and in a long term freezer set at −80 ◦ C for 30 days. It is noteworthy that no significant degradation of nuciferine was observed under the above conditions. 3.2.6. Carry-over and dilution No detectable carry-over was observed in the current LC–MS/MS method. The results of the dilution integrity experiments indicate that the accuracy was within ±13.0% and the precision was less than 6.9%, which could meet the requirements of dilution analysis. 3.3. Pharmacokinetic study The validated method for the quantitation of nuciferine in rat plasma was developed with a low LLOQ of 0.1 ng/ml and applied to a pharmacokinetic study in rats after oral administration of 2.0, 5.0 and 10.0 mg/kg nuciferine and IV administration of 0.2 mg/kg nuciferine. The mean plasma concentration-time curves are shown in Fig. 4. The one-compartment model provided the best fit due to its lower AIC than the two-compartment model, indicating that the former model is more suitable to describe the pharmacokinetic
Fig. 4. Mean plasma concentration-time curve of nuciferine after oral administration (A) of 2 mg/kg, 5 mg/kg, 10 mg/kg nuciferine or intravenous administration (B) of 0.2 mg/kg nuciferine in rats. (n = 6, mean ± SD).
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
27
Table 4 Stability of nuciferine in rat plasma and tissues sample (n = 5). Autosampler (24 h, 4 ◦ C)
Three freeze/thaw cycles
Room temperature (12 h)
Long term (30 days, −80 ◦ C)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
Precision (%)
Accuracy (%)
−5.8 −3.1 4.7
10.0 7.5 9.6
1.2 −3.3 3.7
9.6 8.0 8.1
−8.2 −3.0 6.3
10.9 8.3 10.5
−7.5 −4.3 −1.9
6.0 −2.1 −1.8
11.7 10.0 12.6
2.1 −2.2 −4.5
9.9 7.3 7.3
2.6 −5.4 −5.8
10.9 6.4 12.1
−0.5 −2.7 −3.1
6.8 6.3 5.7
2.8 −2.2 −9.0
8.5 8.2 6.7
1.2 1.4 −7.5
9.1 9.2 9.7
−4.1 3.2 −2.2
7.7 7.3 10.8
−1.6 −2.9 −4.5
0.25 2.5 40
6.7 5.2 10.2
8.4 −5.3 3.4
4.9 12.1 6.9
5.6 −0.5 −4.4
9.4 5.4 9.4
−4.2 1.3 −1.6
6.2 6.3 9.1
−2.5 −3.2 −6.8
Lung
0.25 2.5 80
9.9 7.4 8.2
−3.3 −0.5 −1.7
8.9 5.9 5.3
−5.1 −3.7 −3.5
6.5 6.1 2.9
1.4 0.5 −1.0
4.6 7.2 3.0
−2.0 −2.9 −0.2
Kidney
0.25 2.5 40
9.9 8.1 10.6
−2.8 9.1 9.1
6.8 8.6 9.4
−2.2 1.4 7.2
9.5 7.9 9.3
0.6 4.8 6.0
5.7 7.1 8.0
−2.2 1.1 −3.2
Brain
0.25 2.5 40
10.1 4.5 7.6
0.6 2.8 2.5
5.4 8.0 12.4
6.0 3.3 4.4
9.2 11.4 12.9
5.1 1.3 2.7
5.1 7.6 7.2
−3.6 −3.0 0.9
0.25 1 8
9.0 11.7 8.0
−4.9 3.2 10.0
7.1 9.7 9.3
−4.0 2.5 8.0
7.3 5.1 11.5
−4.5 1.1 8.4
6.1 11.1 11.5
−3.5 3.4 3.1
Biological fluids
Nominal concentration (ng/ml)
Plasma
0.25 2.5 80
5.6 8.0 9.0
Heart
0.25 1 8
6.4 8.8 8.6
Liver
0.25 2.5 40
Spleen
Adipose
administration, Cmax , AUC0–24 h and AUC0–∞ versus the nuciferine dose profiles were linearly with a correlation coefficients of 0.979, 0.985 and 0.988 respectively, showing dose-dependent pharmacokinetics. There was no statistically significant difference in Tmax , t1/2 , Cl and Vd (two tailed paired test, p > 0.05). The value of Tmax and t1/2 indicated that nuciferine was rapidly distributed. The apparent volume of distribution (Vd ) implied that nuciferine exhibited a obvious tissue uptake after oral administration. The values of absolute bioavailability of nuciferine were (3.8 ± 1.4)%, (4.2 ± 1.3)% and (3.9 ± 1.0)% for oral doses of 2.0, 5.0 and 10.0 mg/kg, respectively. These results suggested that its bioavailability was independent of the doses. It was also concluded that nuciferine might have a low absolute bioavailability. Our previous study on absorption profiles of nuciferine in rat intestine [25] showed that the absorption rate constant in the duodenum, jejunum, ileum and colon was 0.014, 0.020, 0.022 and 0.019 min−1 , respectively, indicating that nuciferine had bad absorption from the gastrointestinal tract in rats. Thus, the low oral
bioavailability can be rationalized. Thus, nanoparticle drug delivery system was further investigated to enhance pharmacological activities of nuciferine after oral administration.
3.4. Tissue distribution study Nuciferine distributions in the heart, liver, spleen, lung, kidney, brain and adipose tissue are listed in Fig. 5. The highest concentration was observed at 0.5 h in the lung, followed by the kidney, spleen, brain, liver, adipose tissue and heart after a single dose of IV administration of 0.2 mg/kg nuciferine. Compared with the concentration in plasma, nuciferine could be detected in various tissue samples, indicating that it had a relatively high degree of tissue distribution, which is consistent with the result of the pharmacokinetic study. The sample collected 12 h after administration showed that nuciferine was cleared gradually and no accumulation was observed in the tissues. The higher concentration in the lung,
Table 5 One-compartmental pharmacokinetic parameters of nuciferine after oral administration of 2, 5, 10 mg/kg nuciferine or intravenous administration of 0.2 mg/kg nuciferine in rats (n = 6, mean ± SD). Pharmacokinetic parameters
Unit
Value Oral administration
2 mg/kg t1/2 Vd Cl Tmax Cmax AUC0–24 h AUC0–∞ Bioavailability
h L/kg L/h/kg h ng/ml ng/ml × h ng/ml × h %
1.5 112.4 55.5 0.9 13.5 41.7 41.7 3.8
± ± ± ± ± ± ± ±
Intravenous administration 5 mg/kg
0.8 57.9 22.0 0.4 5.1 19.1 19.1 1.4
0.8 57.3 58.6 0.6 63.4 118.2 118.2 4.2
± ± ± ± ± ± ± ±
10 mg/kg 0.4 24.3 39.9 0.2 19.0 52.5 52.5 1.3
1.0 64.1 52.5 0.5 103.0 207.1 207.5 3.9
± ± ± ± ± ± ± ±
0.6 27.1 15.6 0.1 15.7 67.5 68.3 1.0
0.2 mg/kg 0.6 ± 1.5 ± 1.8 ± – – 119.0 ± 119.0 ± –
0.2 0.5 0.5
31.4 31.4
28
S. Gu et al. / J. Chromatogr. B 961 (2014) 20–28
reformation and the hypothalamus pathway-mediated antiobesity effect of nuciferine. Acknowledgements This study was supported by the National Science and Technology Major Projects of the Twelfth Five-year Plan (No. 2011ZX09302-007-02), Research Fund for the Doctoral Program of Higher Education of China (No. 20110073120097), and the Research Fund of Shanghai JiaoTong University School of Medicine (No. JYY0906). References
Fig. 5. Mean concentration of nuciferine in various tissues at 30 min, 2 h, 6 h and 12 h after an intravenous administration of 0.2 mg/kg nuciferine (n = 6, mean ± SD).
kidney and spleen might be attributed to the high blood flow in these organs. A previous study [13] showed that 80% cholesterol ester associated with lipoproteins was produced by the liver, and nuciferine could clearly suppress HFD-induced alterations in the expression of genes involved in lipid metabolism in the liver to treat dyslipidemia. Siegner et al. [5] also found that adipocytes incubated with the lotus leaf extract with the major constituent of nuciferine exhibited a significant increase in lipolysis-activity and a decrease in adipocyte differentiation capacity, confirming that the liver and adipose tissue were the targets of nuciferine to exert the antiobesity and hypolipidemic effects. The result showed that nuciferine was distributed into the liver and adipose tissue, which provides the material basis for its pharmacological actions. The concentration of nuciferine in the brain tissue was almost equal to that in plasma, suggesting that nuciferine could cross the blood–brain barrier. This finding was potentially important, knowing that the hypothalamus controls the appetite through the feeding center of the brain [26]. As the hypothalamus may be another potential target for the anti-obesity effect of nuciferine, it could be a new field of future research. 4. Conclusion A LC–MS/MS method for the quantification of nuciferine in rat plasma and tissue samples has been established and successfully used to evaluate the pharmacokinetic profile and tissue distribution after oral and IV administration. The method showed high sensitivity, specificity and reliability with an LLOQ of 0.1 ng/ml. The results obtained from the pharmacokinetic and tissue distribution study suggest that nuciferine distribution in the brain, liver and adipose tissue could conduce to the therapeutic effects, but the low absolute bioavailability may limit its further application as an anti-obesity agent. Our ongoing study would focus on the pharmaceutical
[1] Editorial Committee of the Pharmacopoeia of the People’s Republic of China, Pharmacopoeia of the People’s Republic of China, Beijing, China, 2010. [2] P.K. Mukherjee, D. Mukherjee, A.K. Maji, S. Rai, M. Heinrich, J. Pharm. Pharmacol. 61 (2009) 407–422. [3] H. Du, J.S. You, X. Zhao, J.Y. Park, S.H. Kim, K.J. Chang, J. Biomed. Sci. 17 (2010) S42. [4] Y. Ono, E. Hattori, Y. Fukaya, S. Imai, Y. Ohizumi, J. Ethnopharmacol. 106 (2006) 238–244. [5] R. Siegner, S. Heuser, U. Holtzmann, J. Söhle, A. Schepky, T. Raschke, et al., Nutr. Metab. 7 (2010) 66. [6] X.B. Luo, B. Chen, J.J. Liu, S.Z. Yao, Anal. Chim. Acta 538 (2005) 129–133. [7] S. Nakamura, S. Nakashima, G. Tanabe, Y. Oda, N. Yokota, K. Fujimoto, T. Matsumoto, R. Sakuma, T. Ohta, K. Ogawa, S. Nishida, H. Miki, H. Matsuda, O. Muraoka, M. Yoshikawa, Bioorg. Med. Chem. 21 (2013) 779–787. [8] Y. Kashiwada, A. Aoshima, Y. Ikeshiro, Y.P. Chen, H. Furukawa, M. Itoigawa, T. Fujioka, K. Mihashi, L.M. Cosentino, S.L. Morris-Natschke, K.H. Lee, Bioorg. Med. Chem. 13 (2005) 443–448. [9] J. Boustie, J.L. Stigliani, J. Montanha, M. Amoros, M. Payard, L. Girre, J. Nat. Prod. 61 (1998) 480–484. [10] Z.D. Yang, X. Zhang, J. Du, Z.J. Ma, F. Guo, S. Li, X.J. Yao, Nat. Prod. Res. 26 (2012) 387–392. [11] K.H. Nguyen, T.N. Ta, T.H. Pham, Q.T. Nguyen, H.D. Pham, S. Mishra, B.L. Nyomba, J. Ethnopharmacol. 142 (2012) 488–495. [12] X. Han, W. Wang, C. Wu, S. Liu, L. Wang, Progr. Modern Biomed. 8 (2008) 1628–1630. [13] F. Guo, X. Yang, X. Li, R. Feng, C. Guan, Y. Wang, Y. Li, C. Sun, PLoS ONE 8 (2013) e63770. [14] X.O. Wu, Y. Xiong, China Pharm. 7 (2004) 262–264. [15] Y.X. Wang, B. Liu, R.B. Shi, Zhongguo Zhong Yao Za Zhi 33 (2008) 1713–1716. [16] D.F. Ling, S.L. Peng, J. Anhui Agric. Sci. 36 (2008) 10301–10302. [17] T.C. Do, T.D. Nguyen, H. Tran, H. Stuppner, M. Ganzera, J. Chromatogr. A 1302 (2013) 174–180. [18] S.L. Xie, Y.Y. Wang, Y.C. Tan, P.F. Lu, C.J. Tian, R.K. Dai, J. Guangdong Coll. Pharm. 1 (2010) 17–19. [19] M.X. Lin, G.R. Lin, J.F. Chen, Y.H. Guo, J. Fuzhou Univ. 36 (2008) 303–307. [20] Food and Drug Administration of the United States, Guidance Industry Bioanalytical Method Validation, United States. for http://www.fad.gov/cder/guidance/index.hem (accessed May 2001). [21] H. Zhu, X. Zhang, J. Guan, B. Cui, L. Zhao, X. Zhao, J. Pharm. Biomed. 78–79 (2013) 136–140. [22] B. Ma, X. Li, J. Li, Q. Zhang, Y. Liu, X. Yang, et al., J. Pharm. Biomed. 88 (2014) 191–200. [23] Y. Zhang, M. Huo, J. Zhou, S. Xie, P.K. Solver, Comput. Methods Progr. Biomed. 99 (2010) 306–314. [24] K. Balakumar, C.V. Raghavan, N.T. Selvan, R.H. Prasad, S. Abdu, Colloids Surf. B: Biointerf. 112 (2013) 337–343. [25] X.Y. Li, E.J. Zhou, S.Y. Gu, Y. Xu, G.L. Liu, Chin. Pharm. J. J46 (2011) 1417–1420. [26] M.S. Byerly, R.D. Swanson, N.N. Semsarzadeh, P.S. McCulloh, K. Kwon, S. Aja, T.H. Moran, G.W. Wong, S. Blackshaw, Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (2013) R1085–R1095.
