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Simultaneous determination of 14-thienyl methylene matrine and matrine in rat plasma by high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study
Journal of Chromatography B, 974 (2015) 126–130
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of 14-thienyl methylene matrine and matrine in rat plasma by high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study Minjie Jiang a , Lisheng Wang a,∗ , Weizhe Jiang b , Shulin Huang b a b
School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxue Road, Nanning 530004, China School of Pharmaceutical, Guangxi Medical University, Nanning 530021, China
a r t i c l e
i n f o
Article history: Received 27 July 2014 Accepted 29 October 2014 Available online 6 November 2014 Keywords: LC/MS/MS 14-Thienyl methylene matrine Matrine Pharmacokinetics
a b s t r a c t A rapid, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method (HPLC–MS) has been developed and validated for the simultaneous determination of 14-thienyl methylene matrine (TMM) and matrine (MT) in rat plasma in the present study. The analytes were separated on a C18 column (1.9 m, 2.1 mm × 100 mm) with a security guard C18 column (5 m, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With pseudoephedrine hydrochloride as internal standard, sample pretreatment involved in a one-step protein precipitation with isopropanol:ethyl acetate (v/v, 20:80). The method was linear over the concentration ranges of 5–1000 ng/ml for TMM and 10–2000 ng/ml for MT. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. The proposed method enables unambiguous identification and quantification of TMM and MT in vivo. This was the first report on determination of the TMM and MT in rat plasma after oral administration of TMM. The results provided a meaningful basis for evaluating the clinical applications of the medicine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Matrine (MT, Fig. 1a), a quinolizidine alkaloid isolated from Sophora alopecuroides, Sophora flavescens or Sophora subprostrata in traditional Chinese medicine, has been extensively used in China for the treatment of viral hepatitis, cancer, cardiac and skin diseases [1–3]. But the disadvantages of short in vivo half-life and low oral bioavailability limit its clinical application [4]. To improve its pharmacological properties, 14-thienyl methylene matrine (TMM, Fig. 1b) was prepared, its biological activity is higher than that of MT has been demonstrated in previous study [5]. Therefore, a sensitive and accurate analytical method for the simultaneous determination of TMM and MT is required to support pharmacokinetic (PK) study. Recently, much attention has been paid to the absorption and metabolism of matrine and oxymatrine, and a few papers dealing with their pharmacokinetics and pharmacodynamics have
∗ Corresponding author. Tel.: +86 13768509293; fax: +86 07714528697. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.10.041 1570-0232/© 2014 Elsevier B.V. All rights reserved.
been published [6–14] employing mainly high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS) [6–8]. It ensures high sensitivity for quantification with high degree of specificity at relatively short analytical time without a need for complete chromatographic resolution of analytes. However, as far as we are aware, previous researches only aimed directly at administration of single substances, and no paper was reported on the pharmacokinetic studies of TMM and MT simultaneously. In the present study, a new HPLC–MS/MS method was developed and validated for simultaneous quantification of TMM and MT in rat plasma, suitable for the investigation of their pharmacokinetic profile.
2. Experimental 2.1. Reagents and chemicals MT (99.99% pure, batch number 20031010) was supplied by Shanghai Jiagu Pharmaceuticals (Shanghai, China). TMM (99.8% of purity) was synthesized in Department of Chemistry and Chemical Engineering, Guangxi University (Nanning, China).
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working standard solutions (50 l of TMM and MT) to 100 l of blank plasma giving TMM concentrations of 5, 25, 50, 100, 200, 500 and 1000 ng/ml and MT concentrations of 10, 25, 50, 100, 500, 1000 and 2000 ng/ml. The quality control (QC) samples were prepared at low, middle and high concentrations in the same way. 2.4. Plasma sample preparation
Fig. 1. Chemical structures of MT (A), TMM (B) and PPD (C).
Pseudoephedrine hydrochloride (PPD, Fig. 1c, used as internal standard) with purity of greater than 99% were received from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Isopropanol was purchased from Caledon (Georgetown, Ontario, Canada). Ethyl acetate was the provision of Dikma (Richmond Hill, ON, USA). Methanol and acetonitrile, both HPLC grade, were imported from Sigma–Aldrich Chemicals (St. Louis, MO, USA). Deionized water was purified using an Alpha-Q water purification system (Millipore, Bedford, MA, USA) and was filtered using 0.20 m membranes. 2.2. Instruments and conditions HPLC analysis was performed using the Dionex UltiMate 3000 HPLC equipped with a binary pump, an on-line degasser, an auto-sampler and a column temperature controller. Chromatographic separations were performed on a Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 m particle size) protected by a C18 guard column (10 mm × 2.1 mm, 5 m) at 40 ◦ C. The mobile phase consisted of acetonitrile–0.1% formic acid (65:35, v/v). The flow rate was set at 0.2 ml/min. Aliquots of 2 l were injected into HPLC system for analysis. MS analysis was carried out on a Thermo Scientific TSQ Quantum Access MAX triple stage quadrupole mass spectrometer with an electrospray ionization (ESI) source running in a positive-ionization mode. The typical ion source parameters were: spray voltage: 3500 V; sheath gas pressure (N2 ): 20 units; auxiliary gas pressure (N2 ): 5 units; ion transfer tube temperature: 350 ◦ C; collision gas (Ar): 1.5 mTorr; Q1/Q3 peak resolution: 0.7 Da; scan width: 0.002 Da; samples were analyzed via selective-reaction monitoring (SRM) with monitoring ion pairs at m/z 343 → 123 for TMM, m/z 249 → 148 for MT, and m/z 166 → 133 for IS. The scan dwell time was set at 0.1 s for every channel. All data collected in centroid mode were acquired and processed using Xcalibur 2.2 software (Thermo Fisher Scientific Inc., USA). 2.3. Preparation of standards and quality control samples Stock standard solutions of TMM and MT were prepared by dissolving approximate 10 mg of accurately weighted substance in 100 ml of methanol. And the solutions were then serially diluted with methanol to provide working standard solutions of desired concentrations. The PPD (10.0 mg) was dissolved and diluted with methanol to yield a stock solution with a concentration of 1.0 mg/ml, which was further diluted with methanol yielding an IS working solution at concentration of 5.0 g/ml. All the solutions were stored at 4 ◦ C and brought to room temperature before use. Calibration standards were prepared daily by spiking appropriate
100 l of plasma sample in 1.5 ml labeled microcentrifuge tubes was mixed with 20 l of the working internal standard solution and vortexed for 5 s. Then 800 l of isopropanol:ethyl acetate (v/v, 20:80) was added and vortexed for 1 min. The tubes were subsequently centrifuged at 12,000 × g for 10 min. The organic layer was transferred to another tube and evaporated to dryness at ambient temperature under a gentle stream of nitrogen. The residue was reconstituted in 100 l of mobile phase and 2 l was injected into the HPLC–MS/MS system for analysis. 2.5. Method validation Selectivity was assessed by comparing chromatograms of six different batches of blank rat plasma with the corresponding spiked rat plasma. Linearity was assessed by weighted (1/x2 ) analysis of six different calibration curves. Intra- and inter-day precision (the relative standard deviation, RSD) and accuracy (the relative error, RE) were determined by analysis of low, medium, and high QC samples (n = 6) on three different days. The matrix effect was investigated by comparing the peak areas of analytes in the postextraction spiked blank plasma at low and high concentrations with those of the corresponding standard solutions. The extraction recovery was determined by comparing the mean peak areas of six extracted samples at low, medium, and high QC concentrations with the mean peak areas of spike-after-extraction samples. The stability was assessed by analyzing replicates (n = 6) of low and high QC samples during the sample storage and processing procedures. The freeze–thaw stability was determined after three freeze–thaw cycles. Post-preparation stability was estimated by analyzing QC samples at 24 h at 4 ◦ C. Six aliquots of QC samples were stored at −20 ◦ C for 14 days and at ambient temperature for 4 h to determine long-term and short-term stability, respectively. 2.6. Pharmacokinetic (PK) study in rats Male Sprague–Dawley rats weighing from 250 to 300 g were used for PK study. All animal experiments were performed in accordance with institutional guidelines and were approved by the University Committee on Use and Care of Animals, Guangxi Medical University. The aqueous solutions of TMM and MT were separately administrated to 12 rats by gavage at 10 mg/kg. Serial blood samples (0.5 ml) were obtained at 0, 0.25, 0.5, 0.75, 1, 2, 3, 5, 7, 10, 24, 36 h after oral administration separately. All samples were placed into heparinized tubes. After centrifugation at 12,000 rpm and 4 ◦ C for 10 min, plasma was collected and frozen at −20 ◦ C until analysis. Pharmacokinetic parameters were estimated by non-compartmental model using TopFit 2.0 software package (Thomae, Germany). The elimination half-life (t1/2 ) was 0.693/ke , where ke , the elimination rate constant, was calculated by fitting mean data at four terminal points of the plasma concentration profile with a log-linear regression equation using the least-squares method. The maximum drug plasma concentrations (Cmax ) and time to Cmax (Tmax ) were read directly from the observed data. The area under the plasma concentration–time curve from zero to the time of the final measurable sample (AUC0–t ) was calculated by use of the linear-trapezoidal rule.
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Fig. 2. Precursor ion and product ion spectra of TMM (a), MT (b) and PPD (c).
3. Results and discussion 3.1. Chromatography and mass spectrometry The mobile phase was optimized for sensitivity, speed and peak shapes. At first, various proportions of methanol in water were used but gave very poor chromatography. When acetonitrile and 0.1% formic acid in water (v/v, 35:65) was given employed. Under the final chromatographic conditions, PPD, one of the candidate compounds, showed similar retention and ionization to that of matrine and was adopted as internal standard. In the process of sample pretreatment, protein precipitation with methanol or acetonitrile was tried with priority due to its simplicity and the successful examples reported [15,16]. However, it resulted in poor extraction efficiency for TMM in our studied. Then we used isopropanol:ethyl acetate (v/v, 5:95) to extract samples as previously reported [17], it provided clean extracts and higher recoveries for the analyte and the IS. Instead of two extraction cycles, the addition of isopropanol to ethyl acetate (v/v, 20:80) further increased the recoveries without reducing the cleanliness of extracts. The ESI source provided a better response over the APCI source for the two analytes, especially for TMM. In the precursor ion fullscan spectra, the most abundant ions were protonated molecules [M+H]+ m/z 343, 249 and 166 for TMM, MT and IS, respectively. Parameters such as desolvation temperature, ESI source temperature, capillary and spray voltage, flow rate of desolvation gas and auxiliary gas were optimized to obtain highest intensity of protonated molecules of the two compounds and IS. The product ion scan spectra showed high abundance fragment ions at m/z 123, 148 and 133 for TMM, MT and IS, respectively (Fig. 2). The collision gas pressure and collision energy of collision-induced decomposition (CID) were optimized for maximum response of the fragmentation of the two compounds. Selective reaction monitoring (SRM) using the precursor → product ion transition of m/z 343 → 123 for TMM, m/z 249 → 148 for MT and m/z 166 → 133 for IS, the characterization of the analytes fragments are provided in Fig. 3.
3.2. Method validation 3.2.1. Specificity The extent of interference of endogenous plasma constituents with TMM, MT and IS was assessed by inspection of chromatograms derived from processed blank plasma samples. Typical chromatograms obtained from blank plasma, blank plasma spiked with target compounds and IS, and plasma sample after administration of TMM are presented in Fig. 4(a)–(c). The retention times of MT, PPD (IS) and TMM were approximately 1.29, 1.39 and 1.85 min,
Fig. 3. The characterization of the analytes fragments. TMM (a) m/z 343 → 123, MT; (b) m/z 249 → 148 and PPD; (c) m/z 166 → 133.
respectively. There was no endogenous interference and matrix effect on ionization. 3.2.2. Linearity and LLOQ The calibration curves showed good linearity over the concentration range 10–2000 ng/ml for MT and 5–1000 ng/ml for TMM. The typical calibration plot equations and their correlation coefficients were calculated as follows: MT, y = 1.09 × 10−3 x + 1.52 × 10−1 (r2 = 0.9977); TMM, y = 2.96 × 10−4 x + 3.20 × 10−3 (r2 = 0.9978). In the regression equation y = ax + b, x referred to the concentration of the analytes in serum (ng/ml); y referred to the peak area of the analytes and IS. The LLOQ for TMM and MT were 5 and 10 ng/ml with coefficient of variation 8.25% and 9.63%, respectively. Representative chromatogram at the LLOQ provided in Fig. 4(b), the signal-to-noise ratio (SN) were 97 and 51 for MT and TMM respectively. 3.2.3. Precision and accuracy Intra-day and inter-day precision and accuracy were determined by measuring QC samples at three concentrations as described in Section 2. The intra-day accuracy ranged from −2.25% to 1.03% with R.S.D. less than 4.56% for MT and −1.49% to 0.42% with R.S.D. less than 3.58% for TMM. The inter-day accuracy ranged from −3.17% to 1.25% with R.S.D. less than 6.11% for MT and −1.09% to
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Fig. 4. Representative SRM chromatograms of PPD (IS, I), MT (II) and TMM (III) in rat plasmas: (a) a blank rat plasma sample; (b) a blank rat plasma sample spiked with PPD (1000 ng/ml), MT (10 ng/ml), and TMM (5 ng/ml); (C) a rat plasma sample following 1 h after an oral dose of TMM at 10 mg/kg (calculated as MT) to a Sprague–Dawley rat.
Table 1 Validation of the intra-day and inter-day assays. Analytes
Spiked (ng/ml)
MT
25 100 1000 10 50 500
TMM
Intra-day (n = 6)
Inter-day (n = 3)
Measured (ng/ml)
Accuracy (R.E., %)
Precision (R.S.D., %)
Measured (ng/ml)
Accuracy (R.E., %)
Precision (R.S.D., %)
24.75 99.48 1023.06 9.96 49.95 507.53
1.03 0.52 −2.25 0.42 0.11 −1.49
1.20 1.41 4.56 3.58 2.33 1.63
24.83 98.76 1032.76 9.74 49.78 505.52
0.68 1.25 −3.17 2.63 0.44 −1.09
1.43 0.74 6.11 2.41 2.53 2.16
−2.63% with R.S.D. less than 2.53% for TMM. The results indicated that overall reproducibility of the method was acceptable (Table 1).
tested within the time period under the indicated storage conditions.
3.2.4. Extraction recovery and ionization The mean extraction recoveries determined using six replicates of QC samples at three concentration levels were found to be 86.91 ± 6.30% (R.S.D., 10.2%), 89.13 ± 15.16% (R.S.D., 7.31%), 91.40 ± 3.56% (R.S.D., 5.54%) for MT, 86.12 ± 5.21% (R.S.D., 8.45%), 87.23 ± 5.37% (R.S.D., 7.82%), 85.98 ± 4.14% (R.S.D., 5.96%) for TMM and 95.28 ± 5.36% (R.S.D., 5.60%) for IS. As for ionization, the peak area ratios of the two target compounds and IS after spiking evaporated plasma samples at three concentration levels were found to be concentration levels compared to neat standard solutions ranged from 97.3% to 104.2% for MT and 95.3% to 103.6% for TMM, suggesting that the method was free from matrix effect.
3.3. Pharmacokinetic studies
3.2.5. Stability Stability of TMM and MT in processed samples, after freeze–thaw cycle and long-term cold storage (−20 ◦ C, 14 days) were evaluated and summarized in Table 2. The results suggested that the three analytes was stable for 24 h in autosampler condition after preparation, for 14 days under cold storage and within three freeze–thaw cycles in plasma samples, since there was not any obvious change in the concentrations of MT and TMM in plasma
This validated method was successfully applied to PK studies of TMM and MT following oral administration of TMM and MT to 12 Sprague–Dawley rats at 10 mg/kg (all calculated as MT), respectively. MT and the metabolite of TMM concentration time profile conformed to a two-compartment pharmacokinetic model. Mean plasma concentration–time curve of TMM and MT in single dose study is shown in Fig. 5. The estimated pharmacokinetic parameters are shown in Table 3. Since TMM was rapidly transformed into MT in vivo, its concentration in plasma was low. These two analytes exhibited inconsistent tendency in plasma concentration–time profiles although they had similarity in structure features. The MT (after administration of MT) was rapidly absorbed with peak concentrations occurring at around 1.49 h, and was rapidly eliminated from plasma with t1/2 of only about 7.80 h. These values were not significantly different from those reported by Zhang et al. [17] and Yang et al. [18] after we accounted for the differences in dosage. However, the pharmacokinetic behavior for MT (after administration of TMM) was on the contrary. MT was absorbed with Tmax being 2.26 h, and slowly eliminated as the plasma concentration was still much higher than LLOQ after
Table 2 Summary of stability of MT and TMM in plasma (n = 6). Analyte
Spiked (ng/ml)
Stability Ia Measured
MT
TMM
a b c
25 100 1000 10 50 500
24.82 101.8 1006 9.63 51.88 493.8
± ± ± ± ± ±
0.93 2.53 7.20 0.83 2.69 7.78
Stability IIb R.E. (%)
Measured
−0.72 1.76 0.52 −3.73 3.75 −1.24
24.45 98.21 1018 10.38 53.24 509.6
± ± ± ± ± ±
0.45 5.61 17.9 0.46 1.86 11.65
Stability IIIc R.E. (%)
Measured
−2.2 −1.78 1.87 3.83 6.49 1.91
24.21 99.62 990.4 9.85 51.71 494.1
Refers to the post-preparative stability which was determined by processed samples after storage in auto sampler condition for 24 h. Refers to the freeze and thaw stability which was determined by plasma samples after three freeze–thaw cycles. Refers to the long-term cold storage stability which was determined by plasma samples after 14 days storage at −20 ◦ C.
± ± ± ± ± ±
0.96 3.52 16.6 16.57 1.47 2.84
R.E. (%) −3.17 −0.38 −0.96 −1.43 3.42 −1.17
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Fig. 5. Mean plasma concentration–time profiles of TMM (a, following oral administration of TMM) and MT (b, following oral administration of MT) to 12 rats in the Sprague–Dawley rats.
Table 3 Mean pharmacokinetic parameters for MT in rat plasma (n = 6) after oral administration of TMM or MT. Parameter
Administration of TMM
Cmax (g/L) Tmax (h) AUC0–t (g h/L) AUC0–inf (g h/L) t1/2 (h) ke (1/h) V (L) CL (L/h) MRT0–t (h)
1601 2.26 15,862 18,638 18.84 0.038 3.185 0.123 17.77
± ± ± ± ± ± ± ± ±
205.5 0.25 1376 1026 4.36 0.008 0.8960 0.051 4.47
Administration of MT 1437 1.49 7341 7635 7.80 0.096 3.219 0.315 5.86
± ± ± ± ± ± ± ± ±
137.5 0.20 1195 1224 2.25 0.031 0.8853 0.147 0.78
36 h. It is clear that oral administration of TMM is able to enhance oral bioavailability of MT. Chen [19] evaluate the effect of TMM on the suppression of tumor growth of human nasopharyngeal carcinoma CNE1 and CNE2 xenografts in nude mice. The results show that TMM can inhibit the growth of CNE1 and CNE2 cells in vivo, which was evidently stronger than MT. Hence it could be speculated that TMM as a pro-drug releases the active matrine over time. In support of this notion, our lab’s furthered research of TMM metabolism in rat liver microsomes showed that TMM could be metabolized into MT by CYP450. Nevertheless, a review of data in literature [20,21] showed that TMM was able to suppress NPC cells proliferation and induce apoptosis, indicating that TMM still has a higher biological activity than MT in vitro. Furthermore, additional studies are needed to clarify the mechanisms of TMM. 4. Conclusions A sensitive, simple and rapid HPLC–MS/MS method was developed for the simultaneous analysis of TMM and MT in single does
study in rat plasma. It has been successfully applied to the PK study of TMM and MT in the rat. References [1] M. Liu, X.Y. Liu, J.F. Cheng, China J. Chin. Mater. Med. 28 (2003) 801. [2] Y. Long, X.T. Lin, K.L. Zeng, L. Zhang, Hepatobiliary Pancreat. Dis. Int. 3 (2004) 69. [3] J.Y. Liu, J.H. Hu, Q.G. Zhu, F.Q. Li, J. Wang, H.J. Sun, Int. Immunopharmacol. 7 (2007) 816. [4] W. Liu, H. Liu, Lishizhen Med. Mater. Med. Res. 18 (2007) 908. [5] L.S. Wang, Y.j. You, S.Q. Wang, B.M. Liu, X. Lin, M.S. Cheng, J. Guangxi Univ. 3 (2010) 470–472. [6] Y.Q. Wang, Y.Y. Ma, X.H. Li, F. Qin, X.M. Lu, F.M. Li, Biomed. Chromatogr. 21 (2007) 876–882. [7] S.J. Wang, G.J. Wang, X.T. Li, J.G. Sun, R.L. Ma, L.S. Sheng, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 817 (2005) 319–325. [8] X.L. Wu, T.J. Hang, J.P. Shen, Y.D. Zhang, J. Pharm. Biomed. Anal. 41 (2006) 918–924. [9] Y.H. Wei, X.A. Wu, X.F. Liu, J.Y. Luo, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 843 (2006) 10–14. [10] X. Wang, W. Zhang, L.Y. Fan, B. Hao, A.N. Ma, C.X. Cao, Y.X. Wang, Anal. Chim. Acta 594 (2007) 290–296. [11] X.H. Liu, L. Li, J. Sun, Y.H. Sun, T.H. Zhang, D.W. Chen, Z.G. He, Chromatographia 63 (2006) 483. [12] Y. Li, G. Min, Q. Xue, L. Chen, W. Liu, H. Chen, Biomed. Chromatogr. 18 (2004) 619–624. [13] D.S. Sit, G. Gao, F.C. Law, P.C. Li, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 808 (2004) 209–214. [14] Y. Chen, H.X. Chen, P. Du, F.M. Han, Acta Pharm. Sin. 40 (2005) 740–745. [15] X.L. Wu, T.J. Hang, J.P. Shen, Y.D. Zhang, J. Pharm. Biomed. Anal. 41 (2006) 918. [16] Y.J. Wu, J.J. Chen, Y.Y. Cheng, Anal. Bioanal. Chem. 382 (2005) 1595. [17] X.-L. Zhang, H.-R. Xu, W.-L. Chen, N.-N. Chu, X.-N. Li, G.-Y. Liu, C. Yu, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877 (2009) 3253. [18] Z. Yang, S. Gao, T. Yin, K.H. Kulkarni, Y. Teng, M. You, M. Hu, J. Pharm. Biomed. Anal. 51 (2010) 1120. [19] J. Chen, Anti-nasopharyngeal Carcinoma Effects and Its Mechanisms of 14Thienyl methylene matrine, Guangxi Medical University, 2013. [20] L. Jingyu, T. Anzhou, W. Lisheng, R. Wei, X. Lihong, Z. Hualin, China Medical Herald 8 (2011) 13–15. [21] M. Xie, X. Yi, R. Wang, L. Wang, G. He, M. Zhu, C. Qi, Y. Liu, Y. Ye, S. Tan, A. Tang, Cell. Physiol. Biochem. 33 (2014) 1475.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of 14-thienyl methylene matrine and matrine in rat plasma by high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study Minjie Jiang a , Lisheng Wang a,∗ , Weizhe Jiang b , Shulin Huang b a b
School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxue Road, Nanning 530004, China School of Pharmaceutical, Guangxi Medical University, Nanning 530021, China
a r t i c l e
i n f o
Article history: Received 27 July 2014 Accepted 29 October 2014 Available online 6 November 2014 Keywords: LC/MS/MS 14-Thienyl methylene matrine Matrine Pharmacokinetics
a b s t r a c t A rapid, sensitive and selective high-performance liquid chromatography–tandem mass spectrometric method (HPLC–MS) has been developed and validated for the simultaneous determination of 14-thienyl methylene matrine (TMM) and matrine (MT) in rat plasma in the present study. The analytes were separated on a C18 column (1.9 m, 2.1 mm × 100 mm) with a security guard C18 column (5 m, 2.1 mm × 10 mm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was applied for detection. With pseudoephedrine hydrochloride as internal standard, sample pretreatment involved in a one-step protein precipitation with isopropanol:ethyl acetate (v/v, 20:80). The method was linear over the concentration ranges of 5–1000 ng/ml for TMM and 10–2000 ng/ml for MT. The intra-day and inter-day relative standard deviations (RSD) were less than 15% and the relative errors (RE) were all within 15%. The proposed method enables unambiguous identification and quantification of TMM and MT in vivo. This was the first report on determination of the TMM and MT in rat plasma after oral administration of TMM. The results provided a meaningful basis for evaluating the clinical applications of the medicine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Matrine (MT, Fig. 1a), a quinolizidine alkaloid isolated from Sophora alopecuroides, Sophora flavescens or Sophora subprostrata in traditional Chinese medicine, has been extensively used in China for the treatment of viral hepatitis, cancer, cardiac and skin diseases [1–3]. But the disadvantages of short in vivo half-life and low oral bioavailability limit its clinical application [4]. To improve its pharmacological properties, 14-thienyl methylene matrine (TMM, Fig. 1b) was prepared, its biological activity is higher than that of MT has been demonstrated in previous study [5]. Therefore, a sensitive and accurate analytical method for the simultaneous determination of TMM and MT is required to support pharmacokinetic (PK) study. Recently, much attention has been paid to the absorption and metabolism of matrine and oxymatrine, and a few papers dealing with their pharmacokinetics and pharmacodynamics have
∗ Corresponding author. Tel.: +86 13768509293; fax: +86 07714528697. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.10.041 1570-0232/© 2014 Elsevier B.V. All rights reserved.
been published [6–14] employing mainly high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS) [6–8]. It ensures high sensitivity for quantification with high degree of specificity at relatively short analytical time without a need for complete chromatographic resolution of analytes. However, as far as we are aware, previous researches only aimed directly at administration of single substances, and no paper was reported on the pharmacokinetic studies of TMM and MT simultaneously. In the present study, a new HPLC–MS/MS method was developed and validated for simultaneous quantification of TMM and MT in rat plasma, suitable for the investigation of their pharmacokinetic profile.
2. Experimental 2.1. Reagents and chemicals MT (99.99% pure, batch number 20031010) was supplied by Shanghai Jiagu Pharmaceuticals (Shanghai, China). TMM (99.8% of purity) was synthesized in Department of Chemistry and Chemical Engineering, Guangxi University (Nanning, China).
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working standard solutions (50 l of TMM and MT) to 100 l of blank plasma giving TMM concentrations of 5, 25, 50, 100, 200, 500 and 1000 ng/ml and MT concentrations of 10, 25, 50, 100, 500, 1000 and 2000 ng/ml. The quality control (QC) samples were prepared at low, middle and high concentrations in the same way. 2.4. Plasma sample preparation
Fig. 1. Chemical structures of MT (A), TMM (B) and PPD (C).
Pseudoephedrine hydrochloride (PPD, Fig. 1c, used as internal standard) with purity of greater than 99% were received from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Isopropanol was purchased from Caledon (Georgetown, Ontario, Canada). Ethyl acetate was the provision of Dikma (Richmond Hill, ON, USA). Methanol and acetonitrile, both HPLC grade, were imported from Sigma–Aldrich Chemicals (St. Louis, MO, USA). Deionized water was purified using an Alpha-Q water purification system (Millipore, Bedford, MA, USA) and was filtered using 0.20 m membranes. 2.2. Instruments and conditions HPLC analysis was performed using the Dionex UltiMate 3000 HPLC equipped with a binary pump, an on-line degasser, an auto-sampler and a column temperature controller. Chromatographic separations were performed on a Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 m particle size) protected by a C18 guard column (10 mm × 2.1 mm, 5 m) at 40 ◦ C. The mobile phase consisted of acetonitrile–0.1% formic acid (65:35, v/v). The flow rate was set at 0.2 ml/min. Aliquots of 2 l were injected into HPLC system for analysis. MS analysis was carried out on a Thermo Scientific TSQ Quantum Access MAX triple stage quadrupole mass spectrometer with an electrospray ionization (ESI) source running in a positive-ionization mode. The typical ion source parameters were: spray voltage: 3500 V; sheath gas pressure (N2 ): 20 units; auxiliary gas pressure (N2 ): 5 units; ion transfer tube temperature: 350 ◦ C; collision gas (Ar): 1.5 mTorr; Q1/Q3 peak resolution: 0.7 Da; scan width: 0.002 Da; samples were analyzed via selective-reaction monitoring (SRM) with monitoring ion pairs at m/z 343 → 123 for TMM, m/z 249 → 148 for MT, and m/z 166 → 133 for IS. The scan dwell time was set at 0.1 s for every channel. All data collected in centroid mode were acquired and processed using Xcalibur 2.2 software (Thermo Fisher Scientific Inc., USA). 2.3. Preparation of standards and quality control samples Stock standard solutions of TMM and MT were prepared by dissolving approximate 10 mg of accurately weighted substance in 100 ml of methanol. And the solutions were then serially diluted with methanol to provide working standard solutions of desired concentrations. The PPD (10.0 mg) was dissolved and diluted with methanol to yield a stock solution with a concentration of 1.0 mg/ml, which was further diluted with methanol yielding an IS working solution at concentration of 5.0 g/ml. All the solutions were stored at 4 ◦ C and brought to room temperature before use. Calibration standards were prepared daily by spiking appropriate
100 l of plasma sample in 1.5 ml labeled microcentrifuge tubes was mixed with 20 l of the working internal standard solution and vortexed for 5 s. Then 800 l of isopropanol:ethyl acetate (v/v, 20:80) was added and vortexed for 1 min. The tubes were subsequently centrifuged at 12,000 × g for 10 min. The organic layer was transferred to another tube and evaporated to dryness at ambient temperature under a gentle stream of nitrogen. The residue was reconstituted in 100 l of mobile phase and 2 l was injected into the HPLC–MS/MS system for analysis. 2.5. Method validation Selectivity was assessed by comparing chromatograms of six different batches of blank rat plasma with the corresponding spiked rat plasma. Linearity was assessed by weighted (1/x2 ) analysis of six different calibration curves. Intra- and inter-day precision (the relative standard deviation, RSD) and accuracy (the relative error, RE) were determined by analysis of low, medium, and high QC samples (n = 6) on three different days. The matrix effect was investigated by comparing the peak areas of analytes in the postextraction spiked blank plasma at low and high concentrations with those of the corresponding standard solutions. The extraction recovery was determined by comparing the mean peak areas of six extracted samples at low, medium, and high QC concentrations with the mean peak areas of spike-after-extraction samples. The stability was assessed by analyzing replicates (n = 6) of low and high QC samples during the sample storage and processing procedures. The freeze–thaw stability was determined after three freeze–thaw cycles. Post-preparation stability was estimated by analyzing QC samples at 24 h at 4 ◦ C. Six aliquots of QC samples were stored at −20 ◦ C for 14 days and at ambient temperature for 4 h to determine long-term and short-term stability, respectively. 2.6. Pharmacokinetic (PK) study in rats Male Sprague–Dawley rats weighing from 250 to 300 g were used for PK study. All animal experiments were performed in accordance with institutional guidelines and were approved by the University Committee on Use and Care of Animals, Guangxi Medical University. The aqueous solutions of TMM and MT were separately administrated to 12 rats by gavage at 10 mg/kg. Serial blood samples (0.5 ml) were obtained at 0, 0.25, 0.5, 0.75, 1, 2, 3, 5, 7, 10, 24, 36 h after oral administration separately. All samples were placed into heparinized tubes. After centrifugation at 12,000 rpm and 4 ◦ C for 10 min, plasma was collected and frozen at −20 ◦ C until analysis. Pharmacokinetic parameters were estimated by non-compartmental model using TopFit 2.0 software package (Thomae, Germany). The elimination half-life (t1/2 ) was 0.693/ke , where ke , the elimination rate constant, was calculated by fitting mean data at four terminal points of the plasma concentration profile with a log-linear regression equation using the least-squares method. The maximum drug plasma concentrations (Cmax ) and time to Cmax (Tmax ) were read directly from the observed data. The area under the plasma concentration–time curve from zero to the time of the final measurable sample (AUC0–t ) was calculated by use of the linear-trapezoidal rule.
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Fig. 2. Precursor ion and product ion spectra of TMM (a), MT (b) and PPD (c).
3. Results and discussion 3.1. Chromatography and mass spectrometry The mobile phase was optimized for sensitivity, speed and peak shapes. At first, various proportions of methanol in water were used but gave very poor chromatography. When acetonitrile and 0.1% formic acid in water (v/v, 35:65) was given employed. Under the final chromatographic conditions, PPD, one of the candidate compounds, showed similar retention and ionization to that of matrine and was adopted as internal standard. In the process of sample pretreatment, protein precipitation with methanol or acetonitrile was tried with priority due to its simplicity and the successful examples reported [15,16]. However, it resulted in poor extraction efficiency for TMM in our studied. Then we used isopropanol:ethyl acetate (v/v, 5:95) to extract samples as previously reported [17], it provided clean extracts and higher recoveries for the analyte and the IS. Instead of two extraction cycles, the addition of isopropanol to ethyl acetate (v/v, 20:80) further increased the recoveries without reducing the cleanliness of extracts. The ESI source provided a better response over the APCI source for the two analytes, especially for TMM. In the precursor ion fullscan spectra, the most abundant ions were protonated molecules [M+H]+ m/z 343, 249 and 166 for TMM, MT and IS, respectively. Parameters such as desolvation temperature, ESI source temperature, capillary and spray voltage, flow rate of desolvation gas and auxiliary gas were optimized to obtain highest intensity of protonated molecules of the two compounds and IS. The product ion scan spectra showed high abundance fragment ions at m/z 123, 148 and 133 for TMM, MT and IS, respectively (Fig. 2). The collision gas pressure and collision energy of collision-induced decomposition (CID) were optimized for maximum response of the fragmentation of the two compounds. Selective reaction monitoring (SRM) using the precursor → product ion transition of m/z 343 → 123 for TMM, m/z 249 → 148 for MT and m/z 166 → 133 for IS, the characterization of the analytes fragments are provided in Fig. 3.
3.2. Method validation 3.2.1. Specificity The extent of interference of endogenous plasma constituents with TMM, MT and IS was assessed by inspection of chromatograms derived from processed blank plasma samples. Typical chromatograms obtained from blank plasma, blank plasma spiked with target compounds and IS, and plasma sample after administration of TMM are presented in Fig. 4(a)–(c). The retention times of MT, PPD (IS) and TMM were approximately 1.29, 1.39 and 1.85 min,
Fig. 3. The characterization of the analytes fragments. TMM (a) m/z 343 → 123, MT; (b) m/z 249 → 148 and PPD; (c) m/z 166 → 133.
respectively. There was no endogenous interference and matrix effect on ionization. 3.2.2. Linearity and LLOQ The calibration curves showed good linearity over the concentration range 10–2000 ng/ml for MT and 5–1000 ng/ml for TMM. The typical calibration plot equations and their correlation coefficients were calculated as follows: MT, y = 1.09 × 10−3 x + 1.52 × 10−1 (r2 = 0.9977); TMM, y = 2.96 × 10−4 x + 3.20 × 10−3 (r2 = 0.9978). In the regression equation y = ax + b, x referred to the concentration of the analytes in serum (ng/ml); y referred to the peak area of the analytes and IS. The LLOQ for TMM and MT were 5 and 10 ng/ml with coefficient of variation 8.25% and 9.63%, respectively. Representative chromatogram at the LLOQ provided in Fig. 4(b), the signal-to-noise ratio (SN) were 97 and 51 for MT and TMM respectively. 3.2.3. Precision and accuracy Intra-day and inter-day precision and accuracy were determined by measuring QC samples at three concentrations as described in Section 2. The intra-day accuracy ranged from −2.25% to 1.03% with R.S.D. less than 4.56% for MT and −1.49% to 0.42% with R.S.D. less than 3.58% for TMM. The inter-day accuracy ranged from −3.17% to 1.25% with R.S.D. less than 6.11% for MT and −1.09% to
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Fig. 4. Representative SRM chromatograms of PPD (IS, I), MT (II) and TMM (III) in rat plasmas: (a) a blank rat plasma sample; (b) a blank rat plasma sample spiked with PPD (1000 ng/ml), MT (10 ng/ml), and TMM (5 ng/ml); (C) a rat plasma sample following 1 h after an oral dose of TMM at 10 mg/kg (calculated as MT) to a Sprague–Dawley rat.
Table 1 Validation of the intra-day and inter-day assays. Analytes
Spiked (ng/ml)
MT
25 100 1000 10 50 500
TMM
Intra-day (n = 6)
Inter-day (n = 3)
Measured (ng/ml)
Accuracy (R.E., %)
Precision (R.S.D., %)
Measured (ng/ml)
Accuracy (R.E., %)
Precision (R.S.D., %)
24.75 99.48 1023.06 9.96 49.95 507.53
1.03 0.52 −2.25 0.42 0.11 −1.49
1.20 1.41 4.56 3.58 2.33 1.63
24.83 98.76 1032.76 9.74 49.78 505.52
0.68 1.25 −3.17 2.63 0.44 −1.09
1.43 0.74 6.11 2.41 2.53 2.16
−2.63% with R.S.D. less than 2.53% for TMM. The results indicated that overall reproducibility of the method was acceptable (Table 1).
tested within the time period under the indicated storage conditions.
3.2.4. Extraction recovery and ionization The mean extraction recoveries determined using six replicates of QC samples at three concentration levels were found to be 86.91 ± 6.30% (R.S.D., 10.2%), 89.13 ± 15.16% (R.S.D., 7.31%), 91.40 ± 3.56% (R.S.D., 5.54%) for MT, 86.12 ± 5.21% (R.S.D., 8.45%), 87.23 ± 5.37% (R.S.D., 7.82%), 85.98 ± 4.14% (R.S.D., 5.96%) for TMM and 95.28 ± 5.36% (R.S.D., 5.60%) for IS. As for ionization, the peak area ratios of the two target compounds and IS after spiking evaporated plasma samples at three concentration levels were found to be concentration levels compared to neat standard solutions ranged from 97.3% to 104.2% for MT and 95.3% to 103.6% for TMM, suggesting that the method was free from matrix effect.
3.3. Pharmacokinetic studies
3.2.5. Stability Stability of TMM and MT in processed samples, after freeze–thaw cycle and long-term cold storage (−20 ◦ C, 14 days) were evaluated and summarized in Table 2. The results suggested that the three analytes was stable for 24 h in autosampler condition after preparation, for 14 days under cold storage and within three freeze–thaw cycles in plasma samples, since there was not any obvious change in the concentrations of MT and TMM in plasma
This validated method was successfully applied to PK studies of TMM and MT following oral administration of TMM and MT to 12 Sprague–Dawley rats at 10 mg/kg (all calculated as MT), respectively. MT and the metabolite of TMM concentration time profile conformed to a two-compartment pharmacokinetic model. Mean plasma concentration–time curve of TMM and MT in single dose study is shown in Fig. 5. The estimated pharmacokinetic parameters are shown in Table 3. Since TMM was rapidly transformed into MT in vivo, its concentration in plasma was low. These two analytes exhibited inconsistent tendency in plasma concentration–time profiles although they had similarity in structure features. The MT (after administration of MT) was rapidly absorbed with peak concentrations occurring at around 1.49 h, and was rapidly eliminated from plasma with t1/2 of only about 7.80 h. These values were not significantly different from those reported by Zhang et al. [17] and Yang et al. [18] after we accounted for the differences in dosage. However, the pharmacokinetic behavior for MT (after administration of TMM) was on the contrary. MT was absorbed with Tmax being 2.26 h, and slowly eliminated as the plasma concentration was still much higher than LLOQ after
Table 2 Summary of stability of MT and TMM in plasma (n = 6). Analyte
Spiked (ng/ml)
Stability Ia Measured
MT
TMM
a b c
25 100 1000 10 50 500
24.82 101.8 1006 9.63 51.88 493.8
± ± ± ± ± ±
0.93 2.53 7.20 0.83 2.69 7.78
Stability IIb R.E. (%)
Measured
−0.72 1.76 0.52 −3.73 3.75 −1.24
24.45 98.21 1018 10.38 53.24 509.6
± ± ± ± ± ±
0.45 5.61 17.9 0.46 1.86 11.65
Stability IIIc R.E. (%)
Measured
−2.2 −1.78 1.87 3.83 6.49 1.91
24.21 99.62 990.4 9.85 51.71 494.1
Refers to the post-preparative stability which was determined by processed samples after storage in auto sampler condition for 24 h. Refers to the freeze and thaw stability which was determined by plasma samples after three freeze–thaw cycles. Refers to the long-term cold storage stability which was determined by plasma samples after 14 days storage at −20 ◦ C.
± ± ± ± ± ±
0.96 3.52 16.6 16.57 1.47 2.84
R.E. (%) −3.17 −0.38 −0.96 −1.43 3.42 −1.17
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Fig. 5. Mean plasma concentration–time profiles of TMM (a, following oral administration of TMM) and MT (b, following oral administration of MT) to 12 rats in the Sprague–Dawley rats.
Table 3 Mean pharmacokinetic parameters for MT in rat plasma (n = 6) after oral administration of TMM or MT. Parameter
Administration of TMM
Cmax (g/L) Tmax (h) AUC0–t (g h/L) AUC0–inf (g h/L) t1/2 (h) ke (1/h) V (L) CL (L/h) MRT0–t (h)
1601 2.26 15,862 18,638 18.84 0.038 3.185 0.123 17.77
± ± ± ± ± ± ± ± ±
205.5 0.25 1376 1026 4.36 0.008 0.8960 0.051 4.47
Administration of MT 1437 1.49 7341 7635 7.80 0.096 3.219 0.315 5.86
± ± ± ± ± ± ± ± ±
137.5 0.20 1195 1224 2.25 0.031 0.8853 0.147 0.78
36 h. It is clear that oral administration of TMM is able to enhance oral bioavailability of MT. Chen [19] evaluate the effect of TMM on the suppression of tumor growth of human nasopharyngeal carcinoma CNE1 and CNE2 xenografts in nude mice. The results show that TMM can inhibit the growth of CNE1 and CNE2 cells in vivo, which was evidently stronger than MT. Hence it could be speculated that TMM as a pro-drug releases the active matrine over time. In support of this notion, our lab’s furthered research of TMM metabolism in rat liver microsomes showed that TMM could be metabolized into MT by CYP450. Nevertheless, a review of data in literature [20,21] showed that TMM was able to suppress NPC cells proliferation and induce apoptosis, indicating that TMM still has a higher biological activity than MT in vitro. Furthermore, additional studies are needed to clarify the mechanisms of TMM. 4. Conclusions A sensitive, simple and rapid HPLC–MS/MS method was developed for the simultaneous analysis of TMM and MT in single does
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