Journal of Chromatography B, 986–987 (2015) 31–34
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of kurarinone in rat plasma by UPLC–MS/MS Wei-min Zhang a,∗ , Rui-fang Li b , Jian-fei Qiu b , Zhi-yin Zhang b , Hong-bo Wang b , Lu Bian b , Jia-hui Lei b a b
The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan 471003, PR China Medical College of Henan University of Science and Technology, Luoyang, Henan 471003, PR China
a r t i c l e
i n f o
Article history: Received 19 November 2014 Received in revised form 1 February 2015 Accepted 3 February 2015 Available online 9 February 2015 Keywords: Kurarinone UPLC–MS/MS Rat plasma Pharmacokinetics
a b s t r a c t A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0 → 301.2 for kurarinone and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 20–2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7–107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sophora flavescens Aiton is a species of plant in the genus Sophora and has been used in traditional Oriental medicine for the treatment of viral hepatitis, enteritis, cancer, viral myocarditis, gastrointestinal hemorrhage, colpitis, and skin diseases such as psoriasis and eczema [1–3]. Recently, the roots of Sophora flavescens were identified as a rich source of flavonoids, quinolizidine alkaloids, and triterpenoids. Kurarinone (Fig. 1) is one of prenylated flavonones isolated from the roots of Sophora flavescens that exhibits neuroprotective [4,5], anti-immune [6], anti-cancer [7,8], anti-oxidant [9], anti-tyrosinase, [10,11] and anti-glycosidase [12] activities. Several analytical methods, including HPLC [13,14] and LC–MS [15–17], have been developed for the determination of kurarinone in the plants. However, to our best knowledge, there is no bioanalytical method for determination of kurarinone in biological fluids. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer
∗ Corresponding author. Tel.: +86 13513792468. E-mail address: [email protected] (W.-m. Zhang). http://dx.doi.org/10.1016/j.jchromb.2015.02.005 1570-0232/© 2015 Elsevier B.V. All rights reserved.
suitable references in clinical application. Therefore, to characterize the pharmacokinetic properties of kurarinone, it is very necessary to develop an accurate and selective bioanalytical method for the determination of kurarinone in plasma. Ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been evaluated as a faster and more efficient analytical tool compared with current chromatography [18,19]. In the present study, a UPLC–MS/MS method for the determination of kurarinone using chlorzoxazone as an internal standard (IS) was developed. This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect, and recovery. It has been successfully applied in a pharmacokinetic study conducted in rats. 2. Materials and methods 2.1. Chemicals materials Kurarinone (purity > 98%) was provided by Prof. Ma (Dalian Medical University), who purified this compound from the roots of Sophora flavescens as previously described [20]. Chlorzoxazone (purity > 98%, internal standard, IS) was obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC grade and
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W.-m. Zhang et al. / J. Chromatogr. B 986–987 (2015) 31–34
The tubes were vortex mixed for 1.0 min. After centrifugation at 8000 × g for 10 min, the supernatant organic layer was transferred into a 1.5 mL centrifuge tube and dried under nitrogen stream at 40 ◦ C. The dried residue was reconstituted in 75 L of mobile phase and a 6 L aliquot of this was injected into UPLC–MS/MS system for the analysis. 2.5. Method validation Fig. 1. Mass spectra of kurarinone in scan mode with an ESI (−) source.
purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). 2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid) as follows: 0–0.3 min (30–30% A), 0.3–1.0 min (30–90% A), 1.0–2.0 min (90–90% A), 2.0–2.1 min (90–30% A), 2.1–3.0 min (30–30% A). The flow rate was 0.40 mL/min. The overall run time was 3.0 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 437.0 → 301.2 and m/z 168.1 → 132.1 for kurarinone and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 70 psig; ion spray voltage, 4.0 kV; collision energy, 25 eV and temperature, 350 ◦ C. The system control and data analysis were carried out using MassLynx software (Version 4.1) and processed using TargetLynxTM program. 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of kurarinone that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards were 20, 50, 100, 200, 500, 1000 and 2000 ng/mL for kurarinone in rat plasma. The concentrations of QC samples in plasma were 40, 400, 1600 ng/mL for kurarinone. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (500 ng/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C. 2.4. Sample preparation Before analysis, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 20 L of the IS working solution (500 ng/mL) was added to 200 L of collected plasma sample followed by the addition of 1.0 mL ethyl acetate.
Before using this method to determinate kurarinone in rat plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances [21]. Specificity was determined by analysis of blank rat plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of seven concentrations of kurarinone (20–2000 ng/mL) were separately extracted and assayed on three separate days. The linearity for kurarinone was investigated by weighted (1/x2 ) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. To determine the matrix effect, six different blank rat samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (500 ng/mL) in the same manner. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (40, 400, 1600 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy between −15% and 15%. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (500 ng/mL) similarly. The stabilities in rat plasma were tested by analyzing five replicates of plasma samples at three concentration levels (40, 400, 1600 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 3 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 ◦ C for 24 h. The freeze–thaw stability was evaluated after three complete freeze–thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 21 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE% ≤ ±15%) and precision (RSD% ≤ 15%). 2.6. Application to a pharmacokinetic study Male Sprague-Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Henan University of Science and
W.-m. Zhang et al. / J. Chromatogr. B 986–987 (2015) 31–34
33
Fig. 2. Representative chromatograms of kurarinone and IS in rat plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with kurarinone (100 ng/mL) and IS (500 ng/mL); (C) a rat plasma sample after intravenous administration of 10 mg/kg kurarinone (485 ng/mL).
Technology (Henan, China) used to study the pharmacokinetics of kurarinone. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Henan University of Science and Technology and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.5 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after intravenous administration of kurarinone (10 mg/kg). The samples were immediately centrifuged at 4000 × g for 8 min. The plasma obtained (200 L) was stored at −20 ◦ C until analysis. Plasma kurarinone concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Henan University of Science and Technology, China). 3. Results and discussion
extraction with ethyl acetate is cheap, which makes the method suitable for low-cost clinical study. 3.2. Specificity and matrix effect UPLC–MS/MS chromatogram of the analytes in rat plasma samples were shown in Fig. 2. Compared with chromatogram of blank plasma sample, no interference of endogenous peaks was observed. The matrix effect for kurarinone at concentrations of 40, 400 and 1600 ng/mL were measured to be 107.2%, 103.8% and 94.7% (n = 6), respectively. The ME for IS (500 ng/mL) was 95.6% (n = 6). No apparent matrix effect was found to affect the determination of kurarinone and IS in plasma. As a result, the matrix effect from plasma was negligible in this method. 3.3. Linearity and sensitivity
3.1. Method development and optimization Both the positive and negative ionization modes were investigated and good response was achieved in negative ionization mode. Data from the multiple reaction monitoring mode were considered to obtain better selectivity. The most sensitive mass transition was monitored from m/z 437.0–301.2 for kurarinone and from m/z 168.1–132.1 for the IS. In addition, mobile phase compositions, flow rates and LC columns were evaluated and compared to get good chromatographic behavior and appropriate ionization. Eventually, the mobile phase of acetonitrile/water containing 0.1% formic acid with gradient elution at a flow rate 0.4 mL/min was chosen for the analysis of kurarinone and IS. Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) was selected for the chromatographic separation. This system provided higher resolution, greater baseline stability and higher ionization efficiency. In order to maximize recoveries and facilitate the sample preparation, different sample preparation methods were tested. The recoveries were higher when using chloroform or ethyl acetate as extraction solvent, but low for methanol. Due to interferences, it was better to employ the liquid-liquid extraction using ethyl acetate as extraction solvent. No interference was found in the drug-free rat plasma extracted by ethyl acetate. This one-step
The peak area ratios of kurarinone/IS versus the nominal concentrations of kurarinone showed a good linear relationship over the concentration ranges of 20–2000 ng/mL in rat plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.6743x + 1.0374, where y represents the peak area ratios of kurarinone to the IS and x represents plasma concentrations of analyte, with r = 0.9938. The LLOQ in rat plasma was 20 ng/mL with the RSD and RE of 4.5% and 6.3%, respectively. 3.4. Precision, accuracy and recovery The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations (40, 400, 1600 ng/mL) for each biological matrix. The method was reliable and reproducible since RSD% was below 8.2% and RE% was between −9.0% and 8.5% for all the investigated concentrations of kurarinone in rat plasma. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 77.3% to 85.6% for kurarinone. The recovery of IS (500 ng/mL) in plasma was 84.7%. Assay performance data were presented in Table 1. The above results demonstrated that the
Table 1 Precision, accuracy and recovery for kurarinone of QC samples in rat plasma (n = 6). Concentration (ng/mL)
40 400 1600
RSD%
RE%
Recovery (%)
Intra-day
Inter-day
Intra-day
Inter-day
7.6 3.8 4.6
8.2 6.9 4.8
8.5 6.3 −2.6
−9.0 5.6 7.4
85.6 77.3 81.1
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W.-m. Zhang et al. / J. Chromatogr. B 986–987 (2015) 31–34
Table 2 Summary of stability of kurarinone under various storage conditions (n = 5). Conditions
Concentration (ng/mL)
RSD%
RE%
Room temperature, 3 h
40 400 1600 40
7.6 6.5 3.4 8.6
8.2 −7.4 4.2 9.6
4 ◦ C, 24 h
400 1600 40
5.4 6.3 7.9
−7.2 −4.4 8.5
Three freeze–thaw
400 1600 40
7.4 5.2 11.2
3.6 6.0 −8.4
−20 ◦ C, 21 days
400 1600
4.6 7.7
6.2 9.1
when samples of kurarinone were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, kurarinone in samples were stable at −20 ◦ C for 21 days. 3.6. Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of kurarinone in rat plasma. After intravenous administration of 10 mg/kg kurarinone, the main pharmacokinetic parameters of kurarinone were estimated in six rats. The mean plasma concentration–time curve of kurarinone was displayed in Fig. 3, and the main pharmacokinetic parameters of kurarinone were calculated and are summarized in Table 3. 4. Conclusions An UPLC–MS/MS method for the determination of kurarinone in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of kurarinone level in rat plasma using an UPLC–MS/MS method. The method offered sample preparation with a simple one-step liquid–liquid extraction by ethyl acetate and shorter run time of 3.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of kurarinone in rats. References
Fig. 3. Plasma concentration versus time curves after intravenous administration of 10 mg/kg kurarinone in six rats. Table 3 Pharmacokinetic parameters after intravenous administration of 10 mg/kg kurarinone in six rats. Parameters
Mean ± SD
t1/2 (h) Cmax (ng/mL) CL (L/h kg) AUC0 → 6 (ng/mL h) AUC0 → ∞ (ng/mL h)
1.81 1668.01 6.31 1539.14 1595.30
± ± ± ± ±
1.29 101.04 0.55 91.07 102.38
values were within the acceptable range and the method was accurate and precise. 3.5. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions (Table 2). The RSDs of the mean test responses were within 15% in all stability tests. There was no effect on the quantitation for plasma samples kept at room temperature for 3 h and at 4 ◦ C for 24 h. No significant degradation was observed
[1] M.H. Hong, J.Y. Lee, H. Jung, D.H. Jin, H.Y. Go, J.H. Kim, B.H. Jang, Y.C. Shin, S.G. Ko, Toxicol. In Vitro 23 (2009) 251. [2] N. Yang, B. Liang, K. Srivastava, J. Zeng, J. Zhan, L. Brown, H. Sampson, J. Goldfarb, C. Emala, X.M. Li, Phytochemistry 95 (2013) 259. [3] C.Y. Wang, X.Y. Bai, C.H. Wang, Am. J. Chin. Med. 42 (2014) 543. [4] S.J. Park, K.W. Nam, H.J. Lee, E.Y. Cho, U. Koo, W. Mar, Phytomedicine 16 (2009) 1042. [5] G.S. Jeong, B. Li, D.S. Lee, E. Byun, R.B. An, H.O. Pae, H.T. Chung, K.H. Youn, Y.C. Kim, Biol. Pharmaceut. Bull. 31 (2008) 1964. [6] B.H. Kim, K.M. Na, I. Oh, I.H. Song, Y.S. Lee, J. Shin, T.Y. Kim, Biochem. Pharmacol. 85 (2013) 1134. [7] A. De Naeyer, W. Vanden Berghe, V. Pocock, S. Milligan, G. Haegeman, D. De Keukeleire, J. Nat. Prod. 67 (2004) 1829. [8] O.W. Seo, J.H. Kim, K.S. Lee, K.S. Lee, J.H. Kim, M.H. Won, K.S. Ha, Y.G. Kwon, Y.M. Kim, Exp. Mol. Med. 44 (2012) 653. [9] T.S. Jeong, Y.B. Ryu, H.Y. Kim, M.J. Curtis-Long, S.J. An, J.H. Lee, W.S. Lee, K.H. Park, Biol. Pharmaceut. Bull. 31 (2008) 2097. [10] S.J. Kim, K.H. Son, H.W. Chang, S.S. Kang, H.P. Kim, Biol. Pharmaceut. Bull. 26 (2003) 1348. [11] J.K. Son, J.S. Park, J.A. Kim, Y. Kim, S.R. Chung, S.H. Lee, Planta Med. 69 (2003) 559. [12] J.H. Kim, Y.B. Ryu, N.S. Kang, B.W. Lee, J.S. Heo, I.Y. Jeong, K.H. Park, Biol. Pharmaceut. Bull. 29 (2006) 302. [13] R.X. Tan, J.L. Wolfender, L.X. Zhang, W.G. Ma, N. Fuzzati, A. Marston, K. Hostettmann, Phytochemistry 42 (1996) 1305. [14] C. Zhang, Y. Ma, H.M. Gao, X.Q. Liu, L.M. Chen, Q.W. Zhang, Z.M. Wang, A.P. Li, Zhongguo Zhong Xi Yi Jie He Za Zhi 38 (2013) 3520. [15] C.M. He, Z.H. Cheng, D.F. Chen, Chin. J. Nat. Med. 11 (2013) 690. [16] X.L. Piao, X.S. Piao, S.W. Kim, J.H. Park, H.Y. Kim, S.Q. Cai, Biol. Pharmaceut. Bull. 29 (2006) 1911. [17] L. Zhang, L. Xu, S.S. Xiao, Q.F. Liao, Q. Li, J. Liang, X.H. Chen, K.S. Bi, J. Pharmaceut. Biomed. Anal. 44 (2007) 1019. [18] A. de Villiers, F. Lestremau, R. Szucs, S. Gelebart, F. David, P. Sandra, J. Chromatogr. A 1127 (2006) 60. [19] L. van Haandel, J.F. Stobaugh, Bioanalysis 5 (2013) 3023. [20] Y.Q. Shi, X.L. Xin, Q.P. Yuan, C.Y. Wang, B.J. Zhang, J. Hou, Y. Tian, S. Deng, S.S. Huang, X.C. Ma, J. Asian Nat. Prod. Res. 14 (2012) 1002. [21] S. Jamalapuram, P.K. Vuppala, A.H. Abdelazeem, C.R. McCurdy, B.A. Avery, Biomed. Chromatogr. 27 (2013) 1034.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of kurarinone in rat plasma by UPLC–MS/MS Wei-min Zhang a,∗ , Rui-fang Li b , Jian-fei Qiu b , Zhi-yin Zhang b , Hong-bo Wang b , Lu Bian b , Jia-hui Lei b a b
The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan 471003, PR China Medical College of Henan University of Science and Technology, Luoyang, Henan 471003, PR China
a r t i c l e
i n f o
Article history: Received 19 November 2014 Received in revised form 1 February 2015 Accepted 3 February 2015 Available online 9 February 2015 Keywords: Kurarinone UPLC–MS/MS Rat plasma Pharmacokinetics
a b s t r a c t A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine kurarinone in rat plasma using chlorzoxazone as the internal standard (IS). Sample preparation was accomplished through a liquid–liquid extraction procedure with ethyl acetate to 0.2 mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 437.0 → 301.2 for kurarinone and m/z 168.1 → 132.1 for IS. The linearity of this method was found to be within the concentration range of 20–2000 ng/mL with a lower limit of quantification of 20 ng/mL. Only 3.0 min was needed for an analytical run. The matrix effect was 94.7–107.2% for kurarinone. The intra- and inter-day precision (RSD%) were less than 8.2% and accuracy (RE%) was within ±9.0%. The recovery ranged from 77.3% to 85.6%. Kurarinone was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of kurarinone in rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Sophora flavescens Aiton is a species of plant in the genus Sophora and has been used in traditional Oriental medicine for the treatment of viral hepatitis, enteritis, cancer, viral myocarditis, gastrointestinal hemorrhage, colpitis, and skin diseases such as psoriasis and eczema [1–3]. Recently, the roots of Sophora flavescens were identified as a rich source of flavonoids, quinolizidine alkaloids, and triterpenoids. Kurarinone (Fig. 1) is one of prenylated flavonones isolated from the roots of Sophora flavescens that exhibits neuroprotective [4,5], anti-immune [6], anti-cancer [7,8], anti-oxidant [9], anti-tyrosinase, [10,11] and anti-glycosidase [12] activities. Several analytical methods, including HPLC [13,14] and LC–MS [15–17], have been developed for the determination of kurarinone in the plants. However, to our best knowledge, there is no bioanalytical method for determination of kurarinone in biological fluids. Quantification and pharmacokinetics studies on constituents of Traditional Chinese Medicine in plasma are required to offer
∗ Corresponding author. Tel.: +86 13513792468. E-mail address: [email protected] (W.-m. Zhang). http://dx.doi.org/10.1016/j.jchromb.2015.02.005 1570-0232/© 2015 Elsevier B.V. All rights reserved.
suitable references in clinical application. Therefore, to characterize the pharmacokinetic properties of kurarinone, it is very necessary to develop an accurate and selective bioanalytical method for the determination of kurarinone in plasma. Ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) has been evaluated as a faster and more efficient analytical tool compared with current chromatography [18,19]. In the present study, a UPLC–MS/MS method for the determination of kurarinone using chlorzoxazone as an internal standard (IS) was developed. This new method has been fully validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect, and recovery. It has been successfully applied in a pharmacokinetic study conducted in rats. 2. Materials and methods 2.1. Chemicals materials Kurarinone (purity > 98%) was provided by Prof. Ma (Dalian Medical University), who purified this compound from the roots of Sophora flavescens as previously described [20]. Chlorzoxazone (purity > 98%, internal standard, IS) was obtained from Sigma (St. Louis, MO, USA). Acetonitrile and methanol were HPLC grade and
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The tubes were vortex mixed for 1.0 min. After centrifugation at 8000 × g for 10 min, the supernatant organic layer was transferred into a 1.5 mL centrifuge tube and dried under nitrogen stream at 40 ◦ C. The dried residue was reconstituted in 75 L of mobile phase and a 6 L aliquot of this was injected into UPLC–MS/MS system for the analysis. 2.5. Method validation Fig. 1. Mass spectra of kurarinone in scan mode with an ESI (−) source.
purchased from Merck Company (Darmstadt, Germany). HPLC grade water was obtained using a Milli Q system (Millipore, Bedford, USA). 2.2. UPLC–MS/MS conditions Liquid chromatography was performed on an Acquity ultra performance liquid chromatography (UPLC) unit (Waters Corp., Milford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and inline 0.2 m stainless steel frit filter (Waters Corp., Milford, USA). A gradient elution program was conducted for chromatographic separation with mobile phase A (acetonitrile), and mobile phase B (0.1% formic acid) as follows: 0–0.3 min (30–30% A), 0.3–1.0 min (30–90% A), 1.0–2.0 min (90–90% A), 2.0–2.1 min (90–30% A), 2.1–3.0 min (30–30% A). The flow rate was 0.40 mL/min. The overall run time was 3.0 min. An AB Sciex QTRAP 5500 triple quadrupole mass spectrometer equipped with an electro-spray ionization (ESI) source (Toronto, Canada) was used for mass spectrometric detection. The detection was operated in the multiple reaction monitoring (MRM) mode under unit mass resolution (0.7 amu) in the mass analyzers. The dwell time was set to 250 ms for each MRM transition. The MRM transitions were m/z 437.0 → 301.2 and m/z 168.1 → 132.1 for kurarinone and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 50 psig; turbo gas, 70 psig; ion spray voltage, 4.0 kV; collision energy, 25 eV and temperature, 350 ◦ C. The system control and data analysis were carried out using MassLynx software (Version 4.1) and processed using TargetLynxTM program. 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of kurarinone that was used to make the calibration standards and quality control (QC) samples was prepared by dissolving 10 mg in 10 mL methanol to obtain a concentration of 1.00 mg/mL. The stock solution was further diluted with methanol to obtain working solutions at several concentration levels. Calibration standards and QC samples in plasma were prepared by diluting the corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards were 20, 50, 100, 200, 500, 1000 and 2000 ng/mL for kurarinone in rat plasma. The concentrations of QC samples in plasma were 40, 400, 1600 ng/mL for kurarinone. IS stock solution was made at an initial concentration of 1 mg/mL. The IS working solution (500 ng/mL) was made from the stock solution using methanol for dilution. All stock solutions, working solutions, calibration standards and QCs were immediately stored at −20 ◦ C. 2.4. Sample preparation Before analysis, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 20 L of the IS working solution (500 ng/mL) was added to 200 L of collected plasma sample followed by the addition of 1.0 mL ethyl acetate.
Before using this method to determinate kurarinone in rat plasma, the method was fully validated for specificity, linearity, precision, accuracy, recovery, matrix effect and stability according the United States Food and Drug Administration bioanalytical method validation guidances [21]. Specificity was determined by analysis of blank rat plasma samples from six different volunteers, every blank sample was handled by the procedure described in “Sample preparation” and confirmed that endogenous substances did not have the possible interference with the analyte and the IS. To evaluate the linearity, calibration standards of seven concentrations of kurarinone (20–2000 ng/mL) were separately extracted and assayed on three separate days. The linearity for kurarinone was investigated by weighted (1/x2 ) least-squares linear regression of peak area ratios against concentrations. The sensitivity of the method was determined by quantifying the lower limit of quantification (LLOQ). The LLOQ was defined as the lowest acceptable point in the calibration curve which were determined at an acceptable precision and accuracy. To determine the matrix effect, six different blank rat samples were utilized to prepare QC samples and used for assessing the lot-to-lot matrix effect. Matrix effect was evaluated at three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of the pure standard solutions at the same concentrations. The matrix effect of IS was evaluated at the working concentration (500 ng/mL) in the same manner. The precision and accuracy of the method were assessed by determination of QC samples in plasma at different concentrations (40, 400, 1600 ng/mL) on three separate days. Precision was expressed as % relative standard deviation (RSD) and accuracy was expressed as % relative error (RE) between the measured and nominal value. The precision for QC samples was within 15%, and accuracy between −15% and 15%. Extraction recovery experiments which showed an ability to extract the analyte from the test biological samples, were evaluated by comparing the peak areas obtained from extracted QC samples with non-processed standard solutions at three concentrations at the same concentration. Recovery of IS was determined at the working concentration (500 ng/mL) similarly. The stabilities in rat plasma were tested by analyzing five replicates of plasma samples at three concentration levels (40, 400, 1600 ng/mL) in different conditions. The short-term stability was determined after the exposure of the spiked samples at room temperature for 3 h, and the ready-to-inject samples (after extraction) in the autosampler at 4 ◦ C for 24 h. The freeze–thaw stability was evaluated after three complete freeze–thaw cycles (−20 to 25 ◦ C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at −20 ◦ C for 21 days. Samples were considered to be stable if assay values were within the acceptable limits of accuracy (RE% ≤ ±15%) and precision (RSD% ≤ 15%). 2.6. Application to a pharmacokinetic study Male Sprague-Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Henan University of Science and
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Fig. 2. Representative chromatograms of kurarinone and IS in rat plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with kurarinone (100 ng/mL) and IS (500 ng/mL); (C) a rat plasma sample after intravenous administration of 10 mg/kg kurarinone (485 ng/mL).
Technology (Henan, China) used to study the pharmacokinetics of kurarinone. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Henan University of Science and Technology and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.5 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h after intravenous administration of kurarinone (10 mg/kg). The samples were immediately centrifuged at 4000 × g for 8 min. The plasma obtained (200 L) was stored at −20 ◦ C until analysis. Plasma kurarinone concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Henan University of Science and Technology, China). 3. Results and discussion
extraction with ethyl acetate is cheap, which makes the method suitable for low-cost clinical study. 3.2. Specificity and matrix effect UPLC–MS/MS chromatogram of the analytes in rat plasma samples were shown in Fig. 2. Compared with chromatogram of blank plasma sample, no interference of endogenous peaks was observed. The matrix effect for kurarinone at concentrations of 40, 400 and 1600 ng/mL were measured to be 107.2%, 103.8% and 94.7% (n = 6), respectively. The ME for IS (500 ng/mL) was 95.6% (n = 6). No apparent matrix effect was found to affect the determination of kurarinone and IS in plasma. As a result, the matrix effect from plasma was negligible in this method. 3.3. Linearity and sensitivity
3.1. Method development and optimization Both the positive and negative ionization modes were investigated and good response was achieved in negative ionization mode. Data from the multiple reaction monitoring mode were considered to obtain better selectivity. The most sensitive mass transition was monitored from m/z 437.0–301.2 for kurarinone and from m/z 168.1–132.1 for the IS. In addition, mobile phase compositions, flow rates and LC columns were evaluated and compared to get good chromatographic behavior and appropriate ionization. Eventually, the mobile phase of acetonitrile/water containing 0.1% formic acid with gradient elution at a flow rate 0.4 mL/min was chosen for the analysis of kurarinone and IS. Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) was selected for the chromatographic separation. This system provided higher resolution, greater baseline stability and higher ionization efficiency. In order to maximize recoveries and facilitate the sample preparation, different sample preparation methods were tested. The recoveries were higher when using chloroform or ethyl acetate as extraction solvent, but low for methanol. Due to interferences, it was better to employ the liquid-liquid extraction using ethyl acetate as extraction solvent. No interference was found in the drug-free rat plasma extracted by ethyl acetate. This one-step
The peak area ratios of kurarinone/IS versus the nominal concentrations of kurarinone showed a good linear relationship over the concentration ranges of 20–2000 ng/mL in rat plasma. A typical regression equation for the calibration curve resulted in an equation of the line of: y = 0.6743x + 1.0374, where y represents the peak area ratios of kurarinone to the IS and x represents plasma concentrations of analyte, with r = 0.9938. The LLOQ in rat plasma was 20 ng/mL with the RSD and RE of 4.5% and 6.3%, respectively. 3.4. Precision, accuracy and recovery The intra- and inter-day precision and accuracy of the method were determined from the analysis of QC samples at three different concentrations (40, 400, 1600 ng/mL) for each biological matrix. The method was reliable and reproducible since RSD% was below 8.2% and RE% was between −9.0% and 8.5% for all the investigated concentrations of kurarinone in rat plasma. The recovery was calculated by comparing the mean peak areas of the analyte spiked before extraction divided by the areas of analytes samples spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 77.3% to 85.6% for kurarinone. The recovery of IS (500 ng/mL) in plasma was 84.7%. Assay performance data were presented in Table 1. The above results demonstrated that the
Table 1 Precision, accuracy and recovery for kurarinone of QC samples in rat plasma (n = 6). Concentration (ng/mL)
40 400 1600
RSD%
RE%
Recovery (%)
Intra-day
Inter-day
Intra-day
Inter-day
7.6 3.8 4.6
8.2 6.9 4.8
8.5 6.3 −2.6
−9.0 5.6 7.4
85.6 77.3 81.1
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Table 2 Summary of stability of kurarinone under various storage conditions (n = 5). Conditions
Concentration (ng/mL)
RSD%
RE%
Room temperature, 3 h
40 400 1600 40
7.6 6.5 3.4 8.6
8.2 −7.4 4.2 9.6
4 ◦ C, 24 h
400 1600 40
5.4 6.3 7.9
−7.2 −4.4 8.5
Three freeze–thaw
400 1600 40
7.4 5.2 11.2
3.6 6.0 −8.4
−20 ◦ C, 21 days
400 1600
4.6 7.7
6.2 9.1
when samples of kurarinone were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, kurarinone in samples were stable at −20 ◦ C for 21 days. 3.6. Application of the method in a pharmacokinetic study The method described above was successfully applied to determine the concentration of kurarinone in rat plasma. After intravenous administration of 10 mg/kg kurarinone, the main pharmacokinetic parameters of kurarinone were estimated in six rats. The mean plasma concentration–time curve of kurarinone was displayed in Fig. 3, and the main pharmacokinetic parameters of kurarinone were calculated and are summarized in Table 3. 4. Conclusions An UPLC–MS/MS method for the determination of kurarinone in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of kurarinone level in rat plasma using an UPLC–MS/MS method. The method offered sample preparation with a simple one-step liquid–liquid extraction by ethyl acetate and shorter run time of 3.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of kurarinone in rats. References
Fig. 3. Plasma concentration versus time curves after intravenous administration of 10 mg/kg kurarinone in six rats. Table 3 Pharmacokinetic parameters after intravenous administration of 10 mg/kg kurarinone in six rats. Parameters
Mean ± SD
t1/2 (h) Cmax (ng/mL) CL (L/h kg) AUC0 → 6 (ng/mL h) AUC0 → ∞ (ng/mL h)
1.81 1668.01 6.31 1539.14 1595.30
± ± ± ± ±
1.29 101.04 0.55 91.07 102.38
values were within the acceptable range and the method was accurate and precise. 3.5. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions (Table 2). The RSDs of the mean test responses were within 15% in all stability tests. There was no effect on the quantitation for plasma samples kept at room temperature for 3 h and at 4 ◦ C for 24 h. No significant degradation was observed
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