Journal of Chromatography B, 973 (2014) 33–38

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

A simple and sensitive LC–MS/MS method for determination of miltirone in rat plasma and its application to pharmacokinetic studies Long Guo, Li Duan, Xin Dong, Li-Li Dou, Ping Zhou, E-Hu Liu ∗ , Ping Li ∗ State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24 Tongjia Lane, Nanjing 210009, China

a r t i c l e

i n f o

Article history: Received 28 July 2014 Accepted 3 October 2014 Available online 12 October 2014 Keywords: Miltirone LC–MS/MS Rat plasma Pharmacokinetics Bioavailability

a b s t r a c t A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of miltirone concentration in rat plasma. The cytotoxic activity of miltirone was firstly evaluated by the MTT assay and compared with other tanshinones. Quantification was carried out on an Agilent triple quadrupole LC–MS system using multiple reaction monitoring (MRM) mode in positive mode. After simple protein precipitation with acetonitrile, the chromatographic separation of miltirone was achieved by using a Waters Symmetry C18 analytical column (2.1 mm × 100 mm, 3.5 ␮m) with a mobile phase of acetonitrile (A)–water (B) (75:25, v/v) containing 0.5% formic acid. The monitored transitions were set at m/z 283.1 → 223.1 and m/z 361.0 → 232.9 for miltirone and IS, respectively. The calibration curve was linear over the concentration range of 0.5–200 ng/mL with lower limit of quantification of 0.5 ng/mL. The intra- and inter-day accuracy and precision of miltirone were both within acceptable limits. The developed method was successfully applied to a pharmacokinetic study of following oral administration of 20, 40, 60 mg/kg and an intravenous administration of 0.5 mg/kg to rats. The results indicated that miltirone had linear pharmacokinetic properties within the tested dosage range and was poorly absorbed with an absolute bioavailability of approximately 3.4%. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Salvia miltiorrhiza Bunge (Danshen in Chinese), a well-known traditional Chinese medicinal herb, has attracted particular attention from medicinal chemists and clinicians for its reputed therapeutic effects in the treatment of coronary heart and cerebrovascular diseases [1,2]. Tanshinones, the major lipid-soluble chemical constituents in DanShen, have attracted much attention due to the diverse biological activities such as cardiovascular, antibacterial, anti-immunological, antiallergic, anti-oxidant, antitumor and neuroprotective activities [3]. Miltirone is an active tanshinone compound isolated from DanShen. Earlier studies have revealed that miltirone could increase the duration of the hypnotic effects of chloral hydrate and barbiturates in mice, and has been characterized as a partial agonist of the central benzodiazepine receptor [4,5]. Studies also showed that miltirone exerted a wide variety of activities such as antioxidant, anti-inflammatory, antiplasmodial, antitrypanosomal, and anti-tumor effects [6–11]. During the development of a new drug candidate, it is essential to obtain early information regarding its pharmacokinetic

∗ Corresponding authors. Tel.: +86 25 83271379; fax: +86 25 83271379. E-mail addresses: [email protected] (E.-H. Liu), [email protected] (P. Li). http://dx.doi.org/10.1016/j.jchromb.2014.10.008 1570-0232/© 2014 Elsevier B.V. All rights reserved.

parameters as early as possible [12]. To date, pharmacokinetic studies on tanshinones have been mainly focused on tanshinone IIA and cryptotanshinone [13–16], and there is a lack of information concerning other bioactive diterpene quinones, such as miltirone. A previous study has described the effect of miltirone on the metabolism of model probe substrates of CYP450 enzymes in pooled human liver microsomes [17]. To the best of our knowledge, no reports have been described for the sensitive assay of miltirone in biological fluids or its pharmacokinetic parameters. For further clinical application of miltirone, it is necessary to develop a sensitive method for the determination of miltirone in biological samples and to obtain pharmacokinetic information of miltirone. The LC–MS technique combines the efficient separation capability of LC and great power of high sensitivity and structural characterization of MS, and has been widely applied in identifying and quantifying natural compounds in plants or biological fluids [18–24]. In the present study, we evaluated the cytotoxic activity of miltirone and developed a facile, sensitive and specific method based on the liquid chromatography coupled with tandem mass detection (LC–MS/MS) to determine the miltirone concentration in rat plasma. This method has been fully validated for its specificity, accuracy, precision and sensitivity, and successfully applied to the pharmacokinetics study of miltirone after oral and intravenous administration to rats.

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Fig. 1. Chemical structures of miltirone and fenofibrate (IS).

acetonitrile (A) and water (B) (75:25, v/v) containing 0.5% formic acid at a flow rate of 0.3 mL/min. MS spectra were conducted on an Agilent 6460 triple quadrupole mass spectrometer equipped with electrospray ionization source (Agilent Corporation, MA, USA). Quantification was performed in positive using multiple reaction monitoring (MRM) mode of the transitions m/z 283.1 → 223.1 for miltirone and m/z 361.0 → 232.9 for the IS. The MS conditions were as follows: drying gas temperature, 320 ◦ C; drying gas flow, 13 L/min; sheath gas temperature, 250 ◦ C; sheath gas flow, 5 L/min; nebulizer pressure, 45 psi; capillary voltage, 4000 V. Data acquisition was performed with MassHunter Workstation (Agilent Technologies, USA).

2. Experimental 2.4. Preparation of stock solutions 2.1. Reagents and chemicals Miltirone (Fig. 1) was isolated from S. miltiorrhiza in the author’s laboratory. Its chemical structure was unambiguously identified by comparison of the NMR and MS spectra data with the reported literature. Tanshinones IIA, cryptotanshinone and tanshinone I (Supplementary Fig. 1) were obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). Fenofibrate (internal standard, IS) was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). The purity of the reference compounds was determined to be higher than 98% by high performance liquid chromatography-diode array detection analysis. HPLC grade acetonitrile, methanol and formic acid were purchased from ROE (Newark, New Castle, USA). Ultra-pure water was obtained by a Milli-Q system (Millipore, Bedford, MA, USA). Other reagents and solvents were of analytical grade. 2.2. Cytotoxicity of four tanshinones 2.2.1. Cell lines and culture Human hepatoblastoma cell lines HepG2, human lung carcinoma cell lines A549 and human breast carcinoma cell lines MCF-7 were obtained from the American Type Culture Collection (ATCC, USA). Cells grew in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 ◦ C in a humidified atmosphere with 5% CO2 . 2.2.2. Cell viability assay To measure the cytotoxicity of miltirone, tanshinones IIA, cryptotanshinone and tanshinone I on cell proliferation, HepG2, A549 and MCF-7 cells were seeded into 96 well plates at an initial density of 8 × 103 , 3 × 103 and 5 × 103 cells/well, respectively. After incubated overnight, the cells were subsequently exposed to the various concentrations of tanshinones. After incubation for 48 h, 10 ␮L of MTT (methylthiazolyldiphenyl-tetrazolium bromidewas, 5 mg/mL) was added to each well, and the plate was incubated at 37 ◦ C for another 4 h. The MTT solution was then removed and 150 mL of DMSO was added per well. The absorbance at 490 nm was measured by a microplate reader. The viability activity was calculated from the following equation: cell viability (%) = [(absorbance of test sample − absorbance of blank)/(absorbance of control − absorbance of blank)] × 100.

Stock solutions of miltirone and fenobibrate (IS) were prepared both in methanol at a concentration of 0.1 mg/mL. A series of working standard solutions were obtained by further diluting the stock solutions in methanol. The IS working solution (120 ng/mL) was obtained by diluting the stock solution in methanol. Calibration standards were prepared by spiking the appropriate amounts of the standard solutions into blank plasma to obtain final concentrations levels of 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 ng/mL. The quality control (QC) samples were similarly prepared at concentrations of 1, 15 and 150 ng/mL for the low, medium and high concentration, respectively. All work solutions were stored at −20 ◦ C. 2.5. Sample preparation An aliquot of 100 ␮L plasma and 10 ␮L of IS (120 ng/mL) was added into a 1.5 mL centrifuge tube. The mixture was then precipitated with 300 ␮L methanol. After vortexing for 5 min, the sample was centrifuged at 16,200 × g for 10 min. An aliquot of 5 ␮L of the supernatant was injected into the HPLC–MS/MS system for analysis. 2.6. Method validation The method validation assays were performed according to the United States Food and Drug Administration (FDA) guidelines [25]. 2.6.1. Selectivity Selectivity was investigated by comparing the chromatograms of six different batches of blank rat plasma with the corresponding spiked plasma to exclude interference of endogenous substances and metabolites. 2.6.2. Linearity of calibration curves and lower limits of quantification (LLOQ) The calibration curves were performed with nine concentrations (0.5, 1, 2, 5, 10, 20, 50, 100 and 200 ng/mL). The linearity of each calibration curve was determined by plotting the peak area ratio (y) of analytes to IS versus the nominal concentration (x) of analytes with weighted (1/x2 ) least square linear regression. The lowest plasma level of miltirone on the calibration curves (0.5 ng/mL) was recognized as LLOQ with acceptable accuracy and precision (≤20%).

2.3. Instrument and chromatographic conditions Chromatographic analysis was performed on an Agilent series 1290 HPLC system equipped with a quaternary pump, a degasser, an autosampler and a thermostated column compartment (Agilent Technologies, Palo Alto, CA, USA). Chromatographic separation was carried out at 38 ◦ C on a Waters Symmetry C18 analytical column (2.1 mm × 100 mm, 3.5 ␮m). The mobile phase comprised

2.6.3. Precision and accuracy The intra-day precision and accuracy of the method were assessed by determining the QC samples five times on a single day, and the inter-day precision and accuracy were estimated by determining the QC samples over three consecutive days. Relative standard deviation (RSD) and relative error (RE) were used to express the precision and accuracy, respectively.

L. Guo et al. / J. Chromatogr. B 973 (2014) 33–38

2.6.4. Extraction recovery and matrix effect The extraction recovery was evaluated by comparing peak areas obtained from extracted spiked samples with those of the postextracted spiked samples. The matrix effect was evaluated by comparing the peak areas of the post-extracted spiked QC samples with those of corresponding standard solutions. These procedures were repeated for five replicates at three QC concentration levels of 1, 15 and 150 ng/mL. 2.6.5. Stability Stability was examined by analyzing replicates (n = 3) of the three levels of QC samples under different conditions. Short-term stability was evaluated after the exposure of QC samples to room temperature for 12 h. Post-preparative stability was conducted by reanalyzing the QC samples after 24 h in the autosampler at ambient temperature. For freeze/thaw stability, the plasma samples were determined through three freeze (−80 ◦ C)–thaw (room temperature) cycles. Long-term stability was performed by assaying the plasma samples after storage at −80 ◦ C for 15 days. 2.7. Pharmacokinetic study Twenty clean grade male Sprague-Dawley rats (200 ± 20 g) were obtained from the Sino-British SIPPR/BK Lab Animal Ltd. (Shanghai, China). The rats were maintained on a 12 h light–dark cycle with free access to food and water for a week. Prior to the experiments, the rats were allowed to fast for 12 h but with access to water. The rats were randomly assigned to four groups (five rats per group). Three groups were given miltirone (suspended in Tween 80) at a single oral dose of 20, 40 and 60 mg/kg, and one group received a single dose of miltirone (prepared in Tween 80) of 0.5 mg/kg by intravenous injection via caudal vein. Blood samples (approximately 200 ␮L) were collected from the vein of the eye ground with the rat under ether anesthesia into heparinized centrifuge tubes at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h after oral administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h after intravenous administration. Plasma was separated by centrifugation of the whole blood samples at 1500 × g for 10 min and stored at −80 ◦ C until subsequent analysis. All animal studies in the present study were approved by the Animal Ethics Committee of China Pharmaceutical University. Pharmacokinetic parameters including maximum plasma concentration (Cmax ) and time (Tmax ), elimination half-life (t1/2 ), area under the plasma concentration versus time curve from zero to last sampling time (AUC0–t ) and infinity (AUC0–∞ ), mean residence time (MRT) and plasma clearance (CL) of miltirone were estimated using the DAS Software (version 2.0, China State Drug Administration) by non-compartmental method. Data were expressed as mean ± SD. The oral bioavailability (F) of miltirone was calculated by comparing the respective AUC after oral and intravenous administration according to the following equation: F=

oral AUC × iv dose × 100%. iv AUC × oral dose

3. Results and discussion

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Table 1 Cytotoxicity of four tanshinones against HepG2, A549 and MCF-7 cell lines, data represent IC50 values (␮M) mean ± SD (n = 3). Compound

HepG2

Miltirone Tanshinone IIA Cryptotanshinone Tanshinone I

7.81 18.92 13.69 8.74

± ± ± ±

A549 0.26 1.06 0.61 0.76

8.92 19.47 13.17 11.54

MCF-7 ± ± ± ±

0.87 0.86 0.26 0.14

10.12 20.85 13.27 13.05

± ± ± ±

0.17 0.85 0.62 0.37

values of the tested tanshinones against HepG2, A549 and MCF7 cells. The data demonstrated the rank order of potency of the test tanshinones for the tumor cells was: miltirone > tanshinone I > cryptotanshinone > tanshinone IIA. Generally, miltirone had the most potent cytotoxicity against three different tumor cell lines. 3.2. Method development Sample preparation is a critical step for accurate and reliable LC–MS/MS assays. Protein precipitation and liquid–liquid extraction both are commonly used in sample treatment procedures. With the advantages of simplicity and time saving, protein precipitation with methanol was selected in this work. Fenobibrate was used as the IS in the present study [26], because of its similar molecular polarity, retention and ionization condition with the miltirone. The mobile phase played an important role in achieving good chromatographic behavior (including peak symmetry, sensitivity and short run time) and appropriate ionization. In the experiment, it was observed that acetonitrile can give better peak shape and lower background noise than methanol as the organic phase. The addition of formic acid to the mobile phase can significantly improve the sensitivity and peak symmetry by improving the ionization of miltirone and IS. Finally, acetonitrile–0.5% formic acid water (75:25, v/v) was adopted as the mobile phase. The MRM mode afforded by tandem mass spectrometry has great advantage in reducing interference and enhancing sensitivity over the selected ion monitoring (SIM). Miltirone and IS were firstly characterized by MS/MS scan, and then MS/MS product ions mode was obtained to ascertain their precursor ions and to select product ions for use in MRM mode. The full product scan mass spectra of miltirone and IS and their fragmentation schemes of product ions from the parent ions [M+H]+ are shown in Fig. 2. To get the richest relative abundance of product ions, the parameters for fragmentor voltage (110, 115, 120, 125, 130 and 135 V) and collision energy (10, 13, 15, 20, 23, 25 and 30 V) were optimized. Obtained optimal parameters for the richest abundant product ions are shown in Supplementary Table 1. The MRM transitions selected for the detection of miltirone and finofibrate were m/z 283.0 → 223.1 and m/z 361.0 → 232.9, respectively. The retention times of miltirone and IS were about 3.5 and 3.3 min, respectively. No endogenous peak was observed at the retention time for miltirone and IS in any of the six independent blank plasma extracts batches. The representative LC–MS/MS chromatograms of plasma samples for both compounds are shown in Fig. 3.

3.1. Cytotoxic activities of four tanshinones 3.3. Method validation HepG2, A549 and MCF-7 cells were treated with four bioactive compounds, tanshinone IIA at the concentration of 2.5, 5, 10, 15, 20 and 40 ␮M, miltirone, cryptotanshinone and tanshinone I at the concentration of 2.5, 5, 7.5, 10, 15 and 20 ␮M for 48 h. The results demonstrated that the tanshinones significantly inhibited cell proliferation in a concentration-dependent manner when compared with controls (Supplementary Fig. 2). Table 1 summarizes the IC50

3.3.1. Selectivity In the present study, the selectivity was examined using independent plasma samples from six different rats. As shown in Fig. 3, no obvious interferences were observed in the representative chromatogram of a blank plasma sample at the retention times of the analyte and IS.

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Fig. 2. Product ion spectra of miltirone (A) and fenofibrate (IS) (B).

Fig. 3. Typical MRM chromatograms of blank plasma (A), plasma spiked with miltirone at LLOQ and IS (B), plasma spiked with miltirone (15 ng/mL) and IS (C), and plasma sample obtained 1.5 h after an oral administration of 60 mg/kg miltirone (D).

L. Guo et al. / J. Chromatogr. B 973 (2014) 33–38

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Table 2 Precision and accuracy of miltirone in rat plasma. Spiked concentration (ng/mL)

Intra-day (n = 5)

Inter-day (n = 15)

Measured concentration (ng/mL) 0.5 (LLOQ) 1 15 150

0.49 1.07 14.16 138.02

± ± ± ±

0.03 0.05 0.89 4.90

Precision (RSD, %)

Accuracy (RE, %)

Measured concentration (ng/mL)

5.9 5.1 6.3 3.5

−1.3 7.2 −5.6 −8.0

0.48 1.02 13.74 136.93

Table 3 Matrix effect and extraction recovery of miltirone in rat plasma (n = 5). Spiked concentration (ng/mL)

1 15 150

Matrix effect

± ± ± ±

0.05 0.08 0.82 6.94

Precision (RSD, %)

Accuracy (RE, %)

10.4 7.6 6.0 5.1

−4.4 2.1 −8.4 −8.7

found to be 87.8–90.1% with RSD less than 3.7%, which indicated that the extraction procedure was consistent and reproducible.

Extraction recovery

Mean ± SD (%)

RSD (%)

Mean ± SD (%)

RSD (%)

87.8 ± 3.0 88.0 ± 1.6 90.1 ± 3.4

3.4 1.9 3.7

95.3 ± 7.7 90.4 ± 5.1 96.7 ± 4.5

8.1 5.6 4.6

3.3.5. Stability Results of short-term stability, post-preparative stability, freeze and thaw stability, and long-term stability are shown in Table 4. It was demonstrated that the stability offered by this method was satisfactory with the REs and RSD for all samples within general assay acceptability criteria. This result showed that the samples were stable during the routine analysis for the pharmacokinetic study of miltirone.

3.3.2. Calibration curves and LLOQ Linearity for miltirone was obtained over the concentration range of 0.5–200 ng/mL. A typical calibration curve was y = 0.0231x + 0.00206 (r2 = 0.9948), where y represents the peak area ratios of miltirone to the IS and x represents the plasma concentrations of miltirone. The LLOQ of miltirone in rat plasma was 0.5 ng/mL with the intra- and inter-day RSD less than 10.4%, and the intra- and inter-day accuracy of −1.3% and −4.4% (Table 2), which was sufficient for the pharmacokinetic studies of miltirone in rats. 3.3.3. Precision and accuracy The precision and accuracy data for plasma samples are presented in Table 2. The intra- and inter-day precision values (RSD) were less than 11%, and the accuracy (RE) ranged from −8.7% to 7.2%. The data indicated that the accuracy and precision of the method were satisfactory. 3.3.4. Extraction recovery and matrix effect The matrix effect was examined to assess the possibility of ionization suppression or enhancement. The data for the matrix effect are shown in Table 3. The matrix effect ranged from 90% to 97% for miltirone over the three levels of QC samples. The results indicated no obvious matrix effect was observed. The overall mean recoveries of miltirone in plasma at three different concentration levels were

3.4. Pharmacokinetic study The developed method was applied to the pharmacokinetic study of miltirone after oral administration (20, 40 and 60 mg/kg) and intravenous administration (0.5 mg/kg), respectively. The sensitivity and specificity of the assay were found to be sufficient for accurately characterizing the plasma pharmacokinetics of miltirone in rats. The mean plasma concentration versus time profile of miltirone is depicted in Fig. 4 and the partial main pharmacokinetic parameters are shown in Table 5. The results demonstrated that the plasma concentration of miltirone increased rapidly after oral administration of three dosages and all reached the Cmax within 3.6 h, then the concentration went down with an elimination half-life (t1/2 ) of 2.86–3.74 h and a plasma clearance (CL) between 88.06 L/h/kg to 92.28 L/h/kg. Linear regression analysis on the oral administration data displayed preferable colinearity between Cmax , AUC0–t , AUC0–∞ and dosage. In addition, no significant difference was observed in t1/2 , Tmax , and CL (p > 0.05) at three dosage levels except for t1/2 between the dose of 20 and 60 mg/kg (p < 0.05). The results indicated that miltirone has linear pharmacokinetic properties in rats within the tested dosage range. For a drug administrated by oral route, oral bioavailability is undoubtedly one of the most important pharmacokinetic

Table 4 Stability of miltirone in rat plasma (n = 3). Stability conditions

Spiked concentration (ng/mL)

Measured concentration (ng/mL)

Precision (RSD, %)

Accuracy (RE, %)

Short-term (at room temperature for 4 h)

1 15 150

0.99 ± 0.05 13.65 ± 0.69 135.41 ± 6.44

5.2 5.0 4.8

−0.4 −9.0 −9.7

Autosampler (24 h)

1 15 150

0.95 ± 0.02 13.94 ± 0.70 137.66 ± 5.86

1.8 5.0 4.3

−5.3 −7.1 −8.2

Three freeze/thaw cycles (−80 ◦ C)

1 15 150

0.96 ± 0.07 14.00 ± 0.40 138.99 ± 6.63

7.0 2.8 4.8

−4.4 −6.6 −7.3

Long-term (at −80 ◦ C for 15 days)

1 15 150

0.96 ± 0.03 13.92 ± 0.33 140.25 ± 11.72

3.4 2.4 8.4

−4.3 −7.2 −6.5

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Table 5 Mean pharmacokinetic parameters (mean ± SD) after oral administration of 20, 40, 60 mg/kg and intravenous administration of 0.5 mg/kg to rats (n = 5). Parameters

AUC0–t (ng h/mL) AUC0–∞ (ng h/mL) MRT0–t (h) MRT0–∞ (h) t1/2 (h) Tmax (h) CL (L/h/kg) Cmax (ng/mL) F (%)

Administration mode Oral (20 mg/kg)

Oral (40 mg/kg)

Oral (60 mg/kg)

Intravenous (0.5 mg/kg)

215.68 ± 27.90 219.62 ± 26.57 5.41 ± 0.52 5.90 ± 0.39 3.74 ± 1.46 3.60 ± 1.68 92.28 ± 12.52 40.79 ± 5.67 3.32

453.06 ± 84.74 457.73 ± 87.07 5.24 ± 0.46 5.49 ± 0.68 3.36 ± 1.45 3.60 ± 0.55 89.92 ± 16.74 92.70 ± 13.43 3.46

707.36 ± 164.53 709.45 ± 163.73 5.41 ± 0.71 5.49 ± 0.67 2.86 ± 0.65 3.40 ± 0.55 88.06 ± 19.24 144.90 ± 7.56 3.57

160.49 ± 165.3 ± 1.69 ± 2.13 ± 3.33 ± – 3.13 ± – –

30.44 33.24 0.05 0.36 1.43 0.63

Acknowledgments The authors greatly appreciate financial support from Natural Science Foundation of Jiangsu Province (BK20130652), National Natural Science Foundation of China (No. 81130068), Fundamental Research Funds for the Central Universities (ZD2014YW0033), Talent Work Leading Group of Jiangsu Province (333 High-level Talents Training Project) No. BRA2012128, Program for New Century Excellent Talents in University (NECT-13-1034) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.10.008. References

Fig. 4. Mean plasma concentration–time profile (mean ± SD) of miltirone after oral administration of 20, 40, 60 mg/kg (A) and intravenous administration of 0.5 mg/kg (B) to rats (n = 5).

[1] [2] [3] [4] [5] [6] [7]

parameters. The calculated oral bioavailability (F) of miltirone in the present study were 3.32%, 3.46% and 3.57% at low, middle and high dosage levels, respectively. The miltirone showed a poor bioavailability and it might be due to the poor permeability through the intestinal epithelial membrane and significant firstpass effect in the liver. Furthermore, the metabolites in plasma and wide tissue distribution might be responsible for the low bioavailability. The tissue distribution, metabolite and excretion of miltirone need further investigation.

[8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

4. Conclusion In the present study, a sensitive and selective LC–MS/MS method was developed and validated for the quantification of miltirone, a potent antitumor tanshinone, in rat plasma for the first time. The method provided good linearity with a low LLOQ (0.5 ng/mL) and was successfully applied to pharmacokinetic studies of miltirone after oral and intravenous administration to the rats. The developed method could be easily extended to the pharmacokinetic study of other diterpenoid tanshinones. The metabolites, tissue distribution and excretion of miltirone will be reported in the future.

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MS method for determination of miltirone in rat plasma and its application to pharmacokinetic studies.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of miltirone...
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