Journal of Chromatography B, 990 (2015) 181–184
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of trifolirhizin in rat plasma by UPLC: Application to a pharmacokinetic study Kong-hai Ni a , Zheng-de Wen b , Xin-ce Huang b , Chen-xi Wang c , Tian-tian Ye c , Guo-xin Hu c,∗∗ , Meng-tao Zhou b,∗ a
The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 26 January 2015 Accepted 29 March 2015 Available online 4 April 2015 Keywords: Trifolirhizin Rat plasma Pharmacokinetic UPLC
a b s t r a c t In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography (UPLC) has been developed for the determination of trifolirhizin in rat plasma using pirfenidone as internal standard (IS). After sample preparation by a simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and ultraviolet detection set at a wavelength of 366 nm. The method was linear over the concentration range 25–1000 ng/mL with a lower limit of quantification (LLOQ) of 25 ng/mL. Inter- and intra-day precision (RSD%) were all within 10.2% and the accuracy (RE%) was equal or lower than 9.3%. The recovery was in the range of 78.5–86.4% for trifolirhizin and 87.4% for IS. Stability studies showed that trifolirhizin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administration of trifolirhizin to rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Trifolirhizin (Fig. 1) is a pterocarpan which belongs to a special group of isoflavonoids possessing two contiguous benzofuran and benzopyran rings. It was first isolated from Trifolium pratense L. in 1960 and identified in Sophora flavescens by Yagi and co-workers in 1989 [1,2]. Trifolirhizin is thought to be main pharmacologically active constituent in S. flavescens and responsible for many biological and pharmacological activities, such as hepatoprotective [3], skin-whitening [4], anti-inflammatory [2,5], and anti-tumor [6] effects. There have several analytical methods been published for determination of trifolirhizin in raw materials and related traditional Chinese prescriptions, including HPLC [7–9], LC–MS [10], and LC–MS/MS [11,12]. To the best of our knowledge, the information of pharmacokinetic properties of trifolirhizin in Sophora tonkinensis is readily available [13]. However, the pharmacokinetic properties of the constituent contained in an herbal medicine can be quite
∗ Corresponding author. Tel.: +86 057755578286. ∗∗ Co-corresponding author. E-mail addresses: [email protected] (G.-x. Hu), [email protected] (M.-t. Zhou). http://dx.doi.org/10.1016/j.jchromb.2015.03.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
different from the respective pure compound owing to the presence of multiple components. As mentioned above, although the information of pharmacokinetic properties of trifolirhizin in S. tonkinensis is available, to date pharmacokinetic characteristics of the pure trifolirhizin has not been investigated. Therefore, to characterize the pharmacokinetic properties of trifolirhizin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of trifolirhizin in plasma. As a result of recent advances in analysis techniques, ultraperformance liquid chromatography (UPLC) shows a dramatic enhancement in speed, resolution as well as the sensitivity of analysis, while the mobile phase could be able to run at greater linear velocities as compared to standard HPLC. This technique is considered as a new focal point in field of liquid chromatographic studies [14–16]. Thus, in the present work a highly rugged, selective and rapid UPLC method has been developed and fully validated as per the USFDA guidelines for measurement of trifolirhizin in rat plasma using pirfenidone as internal standard (IS). The method offered a small turnaround time for analysis and high sensitivity for the analyte, and utilized only 100 L rat plasma for sample processing using a simple one-step liquid–liquid extraction by ethyl acetate. The method was free from endogenous matrix interference and was successfully applied to a pharmacokinetic study in rats.
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Fig. 1. The chemical structures of trifolirhizin and IS in the present study: (A) trifolirhizin; (B) pirfenidone (IS).
2. Materials and methods 2.1. Chemicals and reagents Trifolirhizin (purity > 98%) was purchased from Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China). Pirfenidone (internal standard, IS, purity > 98%) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, trifluoroacetic acid and ethyl acetate were of LC grade and were purchased from Merck Company (Darmstadt, Germany). Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, MA, USA), was used throughout the study. 2.2. UPLC conditions UPLC analysis was performed with a Waters (Milford, MA, USA) Acquity UPLC system equipped with a binary solvent manager, sample manager, column-heating compartment, and tunable UV detector. This system was controlled by Waters Empower software. The analytes were separated on a 2.1 mm × 50 mm, 1.7 m particle size, Acquity UPLC BEH C18 column. The isocratic analysis was achieved with a mobile phase of solvent A (water):solvent B (acetonitrile):solvent C (0.1% trifluoroacetic acid) = 52:28:20. The flow rate was 0.30 mL/min and total run time was 2.0 min. The column temperature was set at 40 ◦ C. UV absorbance detection was performed at 310 nm. The sample manager was operated at 20 ◦ C and the sample injection volume was 5 L in full-loop mode. 2.3. Standard solutions, calibration standards and quality control (QC) sample Standard stock solutions of trifolirhizin and pirfenidone (IS) were prepared in methanol at 1 mg/mL. Then the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibrations standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of trifolirhizin in plasma were 50, 100, 250, 500, 750, 1000, 1500 ng/mL, and IS was 25 g/mL, respectively. Low-, mid- and high-level quality control (QC) samples containing 100, 400 and 1200 ng/mL of trifolirhizin, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards and QC samples were stored at −20 ◦ C and were brought to room temperature before analysis. 2.4. Sample preparation Before analysis, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 15 L of the IS working solution (25 g/mL) and 50 L of NaOH (1.0 M) were added
to 100 L of collected plasma sample followed by the addition of 0.5 mL ethyl acetate. 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 50 L of acetonitrile–water (50:50, v/v) and a 5 L aliquot of this was injected into UPLC system for the analysis. 2.5. Method validation The method was validated for specificity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) [17,18]. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. 2.5.1. Specificity The specificity of the method was tested by analyzing six different drug-free plasma samples from six rats. Each blank sample was handled by the procedure described in Section 2.4 and confirmed that endogenous substances did not interfere with the analyte and the internal standard. 2.5.2. Linearity and LLOQ Linear calibration curves (y = a + bx) were generated by plotting the peak area ratio (y) of the analyte to IS versus the nominal concentration (x) of the analyte with weighted (1/x2 ) least square linear regression. The LLOQ was defined as the lowest concentration on the calibration curve. The acceptance criteria for accuracy and precision of calibration curve data were 80–120% of the nominal concentrations and relative standard deviation (RSD) of ±20% of the nominal concentration at the LLOQ, respectively. 2.5.3. Accuracy and precision The precision and accuracy of the method were assessed by assaying QC samples at three different concentrations (100, 400 and 1200 ng/mL). Six replicate of each level were analyzed in 1 day or over three consecutive days. Accuracy was expressed as the percentage of observed value to true value, and precision was expressed as the percentage relative standard deviations (RSD, %). 2.5.4. Extraction recovery The recovery showed an ability to extract the analyte from the test biological samples. Recovery of trifolirhizin by ethyl acetate extraction was determined at three different levels (100, 400 and 1200 ng/mL) (n = 6). The recovery of trifolirhizin was calculated by comparing the analyte observed peak area of spiked QC samples in six replicates with those from the non-processed standard
K.-h. Ni et al. / J. Chromatogr. B 990 (2015) 181–184
183
Fig. 2. Representative chromatograms of (2) trifolirhizin and (1) IS in rat plasma samples. (A) A blank plasma sample; (B) blank plasma sample spiked with trifolirhizin and IS; (C) a rat plasma sample taken 0.5 h after intravenous administration of 10.0 mg/kg trifolirhizin in rats.
solutions at the same concentration. The recovery of IS was determined similarly. 2.5.5. Stability To ensure the reliability of the results with regard to handling and storing of the plasma samples, stability studies were carried out on QC samples at concentrations of 100, 400 and 1200 ng/mL. The protocol for the stability assay comprised freeze–thaw stability, short-term and long-term stability. During the freeze–thaw stability assay, the samples were thawed at the ambient temperature without any assistance, and then kept in the freezer (−20 ◦ C) again for minimum of 12 h before carrying out the next thawing, until accomplished three freeze and thaw cycles. The QC samples stored at room temperature for 24 h were evaluated for short-term stability. The long-term stability was determined by analyzing the QC plasma samples after 31 days of storage of −20 ◦ C. The resulted stabilities for these samples were then compared with those of the freshly prepared samples. 2.6. Application to a pharmacokinetic study Male Sprague–Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of trifolirhizin. All six rats were housed at Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University 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 (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 1.5, and 2 h after intravenous administration of trifolirhizin (10.0 mg/kg). The samples were immediately centrifuged at 4000 × g for 8 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma trifolirhizin concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
During analytical method development, direct protein precipitation with popular precipitating agents produced low recovery with high background noise. Furthermore, despite the high performance of the SPE, it was time-consuming and expensive for the preparation of abundant plasma samples. Thus, liquid–liquid extraction was selected to carry out the quantitative analysis. The sample preparation procedure which consisted of ethyl acetate extraction with the evaporation to dryness following was simple and provided high recovery for both trifolirhizin and IS. Other extraction procedures, such as employing dichloromethane as a comparable extracting agent, were studied and showed low recovery (below 60%) and detectable interference from plasma matrix. Therefore, ethyl acetate was chosen as the appropriate extraction solvent. During the IS method, various compounds were tested to decide on a suitable IS which gave satisfactory validation results of UPLC quantification. Among these, pirfenidone was selected as the IS because of its stability and significantly high recovery. Furthermore, it performed good separation from biomatrix of the plasma sample and trifolirhizin in the assay described. 3.2. Assay validation 3.2.1. Specificity Fig. 2 shows the representative chromatograms of blank plasma, a plasma sample spiked with trifolirhizin and IS and a plasma sample obtained at 0.5 h after intravenous administration of trifolirhizin (10.0 mg/kg). No interference was found in the chromatograms of plasma samples at the retention times of trifolirhizin or IS, which were 1.45 and 1.23 min, respectively, and the total running time for each sample was 2.0 min. 3.2.2. Linearity and LLOQ The assay was linear over the concentration range of 50–1500 ng/mL with a typical calibration curve equation of y = 0.2437x − 0.0125 and correlation coefficient of r2 = 0.9996. The LLOQ of trifolirhizin in rat plasma was found to be 50 ng/mL, which are sufficient for the pharmacokinetics study. The precision and accuracy at LLOQ were 7.6 and 5.4%, respectively.
3. Results and discussion 3.1. Method development and optimization To date, although the pharmacokinetic properties of trifolirhizin in S. tonkinensis is readily available [13], the pharmacokinetic characteristics of the pure trifolirhizin has not been elaborated. Thus, it is very necessary to develop an accurate and selective method for the determination of trifolirhizin in plasma to characterize the information of pharmacokinetic properties of trifolirhizin.
3.2.3. 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 (100, 400 and 1200 ng/mL) for each biological matrix. The method was reliable and reproducible since RSD % was below 8.7% and RE% was between −8.4% and 9.4% for all the investigated concentrations of trifolirhizin 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
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Table 1 Precision, accuracy and recovery for trifolirhizin of QC samples in rat plasma (n = 6). RSD%
100 400 1200
RE%
pharmacokinetic parameters from non-compartment model analysis were summarized in Table 2.
Recovery (%)
Intra-day
Inter-day
Intra-day
Inter-day
7.5 5.4 2.1
8.7 4.3 5.7
−6.4 9.4 3.4
8.5 −8.4 2.3
4. Conclusions 78.5 ± 3.2 86.4 ± 4.6 80.6 ± 3.9
A UPLC method for the determination of trifolirhizin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of trifolirhizin level in rat plasma using an UPLC method. The method offered sample preparation with a simple liquid–liquid extraction by ethyl acetate and shorter run time of 2.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of trifolirhizin in rats. References
Fig. 3. Mean plasma concentration time profile after intravenous administration of 10.0 mg/kg trifolirhizin in six rats.
spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 78.5% to 86.4% for trifolirhizin. The recovery of IS (25 g/mL) in plasma was 87.4%. Assay performance data were presented in Table 1. The above results demonstrated that the values were within the acceptable range and the method was accurate and precise. 3.2.4. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. There was no statistically significant effect on the quantitation for plasma samples kept at room temperature for 24 h. No significant degradation was observed when samples of trifolirhizin were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, trifolirhizin in samples were stable at −20 ◦ C for 31 days. 3.3. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral administration of 10.0 mg/kg trifolirhizin was shown in Fig. 3. The main Table 2 The main pharmacokinetic parameters after oral administration of 10.0 mg/kg trifolirhizin in six rats. Parameters
Trifolirhizin
t1/2 (h) Vd (L/kg) MRT (h) CL (L/h/kg) Cmax (ng/mL) AUC0 → t (ng/mL h) AUC0 → ∞ (ng/mL h)
0.68 16.65 0.65 17.14 1066.83 552.44 599.56
± ± ± ± ± ± ±
0.15 3.84 0.09 3.26 125.70 91.26 101.03
[1] Y. Fujise, T. Toda, S. Ito, Isolation of trifolirhizin from Ononis spinosa L, Chem. Pharm. Bull. 13 (1965) 93. [2] H. Zhou, H. Lutterodt, Z. Cheng, L.L. Yu, Anti-Inflammatory and antiproliferative activities of trifolirhizin, a flavonoid from Sophora flavescens roots, J. Agric. Food Chem. 57 (2009) 4580. [3] M.S. Abdel-Kader, Preliminary pharmacological study of the pterocarpans macckian and trifolirhizin isolated from the roots of Ononis vaginalis, Pak. J. Pharm. Sci. 23 (2010) 182. [4] S.K. Hyun, W.H. Lee, M. Jeong da, Y. Kim, J.S. Choi, Inhibitory effects of kurarinol, kuraridinol, and trifolirhizin from Sophora flavescens on tyrosinase and melanin synthesis, Biol. Pharm. Bull. 31 (2008) 154. [5] N. Yang, B. Liang, K. Srivastava, J. Zeng, J. Zhan, L. Brown, H. Sampson, J. Goldfarb, C. Emala, X.M. Li, The Sophora flavescens flavonoid compound trifolirhizin inhibits acetylcholine induced airway smooth muscle contraction, Phytochemistry 95 (2013) 259. [6] Y. Aratanechemuge, H. Hibasami, H. Katsuzaki, K. Imai, T. Komiya, Induction of apoptosis by maackiain and trifolirhizin (maackiain glycoside) isolated from sanzukon (Sophora Subprostrate Chen et T. Chen) in human promyelotic leukemia HL-60 cells, Oncol. Rep. 12 (2004) 1183. [7] X.C. Ma, X.L. Xin, K.X. Liu, B.J. Zhang, F.Y. Li, D.A. Guo, Simultaneous determination of nine major flavonoids in Sophora flavescens by RP-LC, Chromatographia 68 (2008) 471. [8] H.Y. Ma, W.S. Zhou, F.J. Chu, D. Wang, S.W. Liang, S. Li, [HPLC fingerprint of flavonoids in Sophora flavescens and determination of five components], Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China J. Chin. Mater. Med. 38 (2013) 2690. [9] B. Liu, R.B. Shi, L.J. Zhu, [HPLC fingerprint of flavonoids of Kushen Tang and its correlation to Scutellaria baicalensis and Sophora flavescens], Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China J. Chin. Mater. Med. 32 (2007) 1631. [10] C.M. He, Z.H. Cheng, D.F. Chen, Qualitative and quantitative analysis of flavonoids in Sophora tonkinensis by LC/MS and HPLC, Chin. J. Nat. Med. 11 (2013) 690. [11] H.E. Kohrt, I. Sagiv-Barfi, S. Rafiq, S.E. Herman, J.P. Butchar, C. Cheney, X. Zhang, J.J. Buggy, N. Muthusamy, R. Levy, A.J. Johnson, J.C. Byrd, Ibrutinib antagonizes rituximab-dependent NK cell-mediated cytotoxicity, Blood 123 (2014) 1957. [12] L. Zhang, L. Xu, S.S. Xiao, Q.F. Liao, Q. Li, J. Liang, X.H. Chen, K.S. Bi, Characterization of flavonoids in the extract of Sophora flavescens Ait. by highperformance liquid chromatography coupled with diode-array detector and electrospray ionization mass spectrometry, J. Pharm. Biomed. Anal. 44 (2007) 1019. [13] H. Yoo, K.H. Ryu, S.K. Bae, J. Kim, Simultaneous determination of trifolirhizin, (-)-maackiain, (-)-sophoranone, and 2-(2,4-dihydroxyphenyl)5,6-methylenedioxybenzofuran from Sophora tonkinensis in rat plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study, J. Sep. Sci. 37 (2014) 3235. [14] S. Pieters, B. Dejaegher, Y. Vander Heyden, Emerging analytical separation techniques with high throughput potential for pharmaceutical analysis, part I: Stationary phase and instrumental developments in LC, Comb. Chem. High Throughput Screen. 13 (2010) 510. [15] B. Dejaegher, S. Pieters, Y. Vander Heyden, Emerging analytical separation techniques with high-throughput potential for pharmaceutical analysis, part II: Novel chromatographic modes, Comb. Chem. High Throughput Screen. 13 (2010) 530. [16] A. Kumar, G. Saini, A. Nair, R. Sharma, UPLC: a preeminent technique in pharmaceutical analysis, Acta Pol. Pharm. 69 (2012) 371. [17] P. van Amsterdam, A. Companjen, M. Brudny-Kloeppel, M. Golob, S. Luedtke, P. Timmerman, The European Bioanalysis Forum community’s evaluation, interpretation and implementation of the European Medicines Agency guideline on Bioanalytical Method Validation, Bioanalysis 5 (2013) 645. [18] F. Garofolo, J. Michon, V. Leclaire, B. Booth, S. Lowes, C.T. Viswanathan, J. Welink, S. Haidar, L.D. Teixeira, D. Tang, B. Desilva, US FDA/EMA harmonization of their bioanalytical guidance/guideline and activities of the Global Bioanalytical Consortium, Bioanalysis 4 (2012) 231.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of trifolirhizin in rat plasma by UPLC: Application to a pharmacokinetic study Kong-hai Ni a , Zheng-de Wen b , Xin-ce Huang b , Chen-xi Wang c , Tian-tian Ye c , Guo-xin Hu c,∗∗ , Meng-tao Zhou b,∗ a
The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 26 January 2015 Accepted 29 March 2015 Available online 4 April 2015 Keywords: Trifolirhizin Rat plasma Pharmacokinetic UPLC
a b s t r a c t In this study, a simple, sensitive, and robust analytical method based on ultra-performance liquid chromatography (UPLC) has been developed for the determination of trifolirhizin in rat plasma using pirfenidone as internal standard (IS). After sample preparation by a simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m particle size) and ultraviolet detection set at a wavelength of 366 nm. The method was linear over the concentration range 25–1000 ng/mL with a lower limit of quantification (LLOQ) of 25 ng/mL. Inter- and intra-day precision (RSD%) were all within 10.2% and the accuracy (RE%) was equal or lower than 9.3%. The recovery was in the range of 78.5–86.4% for trifolirhizin and 87.4% for IS. Stability studies showed that trifolirhizin was stable under a variety of storage conditions. The method was successfully applied to a pharmacokinetic study involving oral administration of trifolirhizin to rats. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Trifolirhizin (Fig. 1) is a pterocarpan which belongs to a special group of isoflavonoids possessing two contiguous benzofuran and benzopyran rings. It was first isolated from Trifolium pratense L. in 1960 and identified in Sophora flavescens by Yagi and co-workers in 1989 [1,2]. Trifolirhizin is thought to be main pharmacologically active constituent in S. flavescens and responsible for many biological and pharmacological activities, such as hepatoprotective [3], skin-whitening [4], anti-inflammatory [2,5], and anti-tumor [6] effects. There have several analytical methods been published for determination of trifolirhizin in raw materials and related traditional Chinese prescriptions, including HPLC [7–9], LC–MS [10], and LC–MS/MS [11,12]. To the best of our knowledge, the information of pharmacokinetic properties of trifolirhizin in Sophora tonkinensis is readily available [13]. However, the pharmacokinetic properties of the constituent contained in an herbal medicine can be quite
∗ Corresponding author. Tel.: +86 057755578286. ∗∗ Co-corresponding author. E-mail addresses: [email protected] (G.-x. Hu), [email protected] (M.-t. Zhou). http://dx.doi.org/10.1016/j.jchromb.2015.03.031 1570-0232/© 2015 Elsevier B.V. All rights reserved.
different from the respective pure compound owing to the presence of multiple components. As mentioned above, although the information of pharmacokinetic properties of trifolirhizin in S. tonkinensis is available, to date pharmacokinetic characteristics of the pure trifolirhizin has not been investigated. Therefore, to characterize the pharmacokinetic properties of trifolirhizin, it is very necessary to develop an accurate and selective bioanalytical method for the determination of trifolirhizin in plasma. As a result of recent advances in analysis techniques, ultraperformance liquid chromatography (UPLC) shows a dramatic enhancement in speed, resolution as well as the sensitivity of analysis, while the mobile phase could be able to run at greater linear velocities as compared to standard HPLC. This technique is considered as a new focal point in field of liquid chromatographic studies [14–16]. Thus, in the present work a highly rugged, selective and rapid UPLC method has been developed and fully validated as per the USFDA guidelines for measurement of trifolirhizin in rat plasma using pirfenidone as internal standard (IS). The method offered a small turnaround time for analysis and high sensitivity for the analyte, and utilized only 100 L rat plasma for sample processing using a simple one-step liquid–liquid extraction by ethyl acetate. The method was free from endogenous matrix interference and was successfully applied to a pharmacokinetic study in rats.
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Fig. 1. The chemical structures of trifolirhizin and IS in the present study: (A) trifolirhizin; (B) pirfenidone (IS).
2. Materials and methods 2.1. Chemicals and reagents Trifolirhizin (purity > 98%) was purchased from Chengdu Mansite Pharmaceutical Co. Ltd. (Chengdu, China). Pirfenidone (internal standard, IS, purity > 98%) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, trifluoroacetic acid and ethyl acetate were of LC grade and were purchased from Merck Company (Darmstadt, Germany). Ultrapure water, prepared by a Milli-Q Reagent water system (Millipore, MA, USA), was used throughout the study. 2.2. UPLC conditions UPLC analysis was performed with a Waters (Milford, MA, USA) Acquity UPLC system equipped with a binary solvent manager, sample manager, column-heating compartment, and tunable UV detector. This system was controlled by Waters Empower software. The analytes were separated on a 2.1 mm × 50 mm, 1.7 m particle size, Acquity UPLC BEH C18 column. The isocratic analysis was achieved with a mobile phase of solvent A (water):solvent B (acetonitrile):solvent C (0.1% trifluoroacetic acid) = 52:28:20. The flow rate was 0.30 mL/min and total run time was 2.0 min. The column temperature was set at 40 ◦ C. UV absorbance detection was performed at 310 nm. The sample manager was operated at 20 ◦ C and the sample injection volume was 5 L in full-loop mode. 2.3. Standard solutions, calibration standards and quality control (QC) sample Standard stock solutions of trifolirhizin and pirfenidone (IS) were prepared in methanol at 1 mg/mL. Then the stock solutions were diluted with methanol to obtain fresh standard working solution. Calibrations standards were prepared by adding corresponding working solutions in drug-free plasma. The final concentrations of trifolirhizin in plasma were 50, 100, 250, 500, 750, 1000, 1500 ng/mL, and IS was 25 g/mL, respectively. Low-, mid- and high-level quality control (QC) samples containing 100, 400 and 1200 ng/mL of trifolirhizin, were prepared in a manner similar to that used for the preparation of calibration samples. All stock solutions, working solutions, calibration standards and QC samples were stored at −20 ◦ C and were brought to room temperature before analysis. 2.4. Sample preparation Before analysis, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 15 L of the IS working solution (25 g/mL) and 50 L of NaOH (1.0 M) were added
to 100 L of collected plasma sample followed by the addition of 0.5 mL ethyl acetate. 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 50 L of acetonitrile–water (50:50, v/v) and a 5 L aliquot of this was injected into UPLC system for the analysis. 2.5. Method validation The method was validated for specificity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) [17,18]. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. 2.5.1. Specificity The specificity of the method was tested by analyzing six different drug-free plasma samples from six rats. Each blank sample was handled by the procedure described in Section 2.4 and confirmed that endogenous substances did not interfere with the analyte and the internal standard. 2.5.2. Linearity and LLOQ Linear calibration curves (y = a + bx) were generated by plotting the peak area ratio (y) of the analyte to IS versus the nominal concentration (x) of the analyte with weighted (1/x2 ) least square linear regression. The LLOQ was defined as the lowest concentration on the calibration curve. The acceptance criteria for accuracy and precision of calibration curve data were 80–120% of the nominal concentrations and relative standard deviation (RSD) of ±20% of the nominal concentration at the LLOQ, respectively. 2.5.3. Accuracy and precision The precision and accuracy of the method were assessed by assaying QC samples at three different concentrations (100, 400 and 1200 ng/mL). Six replicate of each level were analyzed in 1 day or over three consecutive days. Accuracy was expressed as the percentage of observed value to true value, and precision was expressed as the percentage relative standard deviations (RSD, %). 2.5.4. Extraction recovery The recovery showed an ability to extract the analyte from the test biological samples. Recovery of trifolirhizin by ethyl acetate extraction was determined at three different levels (100, 400 and 1200 ng/mL) (n = 6). The recovery of trifolirhizin was calculated by comparing the analyte observed peak area of spiked QC samples in six replicates with those from the non-processed standard
K.-h. Ni et al. / J. Chromatogr. B 990 (2015) 181–184
183
Fig. 2. Representative chromatograms of (2) trifolirhizin and (1) IS in rat plasma samples. (A) A blank plasma sample; (B) blank plasma sample spiked with trifolirhizin and IS; (C) a rat plasma sample taken 0.5 h after intravenous administration of 10.0 mg/kg trifolirhizin in rats.
solutions at the same concentration. The recovery of IS was determined similarly. 2.5.5. Stability To ensure the reliability of the results with regard to handling and storing of the plasma samples, stability studies were carried out on QC samples at concentrations of 100, 400 and 1200 ng/mL. The protocol for the stability assay comprised freeze–thaw stability, short-term and long-term stability. During the freeze–thaw stability assay, the samples were thawed at the ambient temperature without any assistance, and then kept in the freezer (−20 ◦ C) again for minimum of 12 h before carrying out the next thawing, until accomplished three freeze and thaw cycles. The QC samples stored at room temperature for 24 h were evaluated for short-term stability. The long-term stability was determined by analyzing the QC plasma samples after 31 days of storage of −20 ◦ C. The resulted stabilities for these samples were then compared with those of the freshly prepared samples. 2.6. Application to a pharmacokinetic study Male Sprague–Dawley rats (180–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of trifolirhizin. All six rats were housed at Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University 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 (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 1.5, and 2 h after intravenous administration of trifolirhizin (10.0 mg/kg). The samples were immediately centrifuged at 4000 × g for 8 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma trifolirhizin concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
During analytical method development, direct protein precipitation with popular precipitating agents produced low recovery with high background noise. Furthermore, despite the high performance of the SPE, it was time-consuming and expensive for the preparation of abundant plasma samples. Thus, liquid–liquid extraction was selected to carry out the quantitative analysis. The sample preparation procedure which consisted of ethyl acetate extraction with the evaporation to dryness following was simple and provided high recovery for both trifolirhizin and IS. Other extraction procedures, such as employing dichloromethane as a comparable extracting agent, were studied and showed low recovery (below 60%) and detectable interference from plasma matrix. Therefore, ethyl acetate was chosen as the appropriate extraction solvent. During the IS method, various compounds were tested to decide on a suitable IS which gave satisfactory validation results of UPLC quantification. Among these, pirfenidone was selected as the IS because of its stability and significantly high recovery. Furthermore, it performed good separation from biomatrix of the plasma sample and trifolirhizin in the assay described. 3.2. Assay validation 3.2.1. Specificity Fig. 2 shows the representative chromatograms of blank plasma, a plasma sample spiked with trifolirhizin and IS and a plasma sample obtained at 0.5 h after intravenous administration of trifolirhizin (10.0 mg/kg). No interference was found in the chromatograms of plasma samples at the retention times of trifolirhizin or IS, which were 1.45 and 1.23 min, respectively, and the total running time for each sample was 2.0 min. 3.2.2. Linearity and LLOQ The assay was linear over the concentration range of 50–1500 ng/mL with a typical calibration curve equation of y = 0.2437x − 0.0125 and correlation coefficient of r2 = 0.9996. The LLOQ of trifolirhizin in rat plasma was found to be 50 ng/mL, which are sufficient for the pharmacokinetics study. The precision and accuracy at LLOQ were 7.6 and 5.4%, respectively.
3. Results and discussion 3.1. Method development and optimization To date, although the pharmacokinetic properties of trifolirhizin in S. tonkinensis is readily available [13], the pharmacokinetic characteristics of the pure trifolirhizin has not been elaborated. Thus, it is very necessary to develop an accurate and selective method for the determination of trifolirhizin in plasma to characterize the information of pharmacokinetic properties of trifolirhizin.
3.2.3. 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 (100, 400 and 1200 ng/mL) for each biological matrix. The method was reliable and reproducible since RSD % was below 8.7% and RE% was between −8.4% and 9.4% for all the investigated concentrations of trifolirhizin 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
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Table 1 Precision, accuracy and recovery for trifolirhizin of QC samples in rat plasma (n = 6). RSD%
100 400 1200
RE%
pharmacokinetic parameters from non-compartment model analysis were summarized in Table 2.
Recovery (%)
Intra-day
Inter-day
Intra-day
Inter-day
7.5 5.4 2.1
8.7 4.3 5.7
−6.4 9.4 3.4
8.5 −8.4 2.3
4. Conclusions 78.5 ± 3.2 86.4 ± 4.6 80.6 ± 3.9
A UPLC method for the determination of trifolirhizin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of trifolirhizin level in rat plasma using an UPLC method. The method offered sample preparation with a simple liquid–liquid extraction by ethyl acetate and shorter run time of 2.0 min. The method meets the requirement of high sample throughput in bioanalysis and has been successfully applied to the pharmacokinetic study of trifolirhizin in rats. References
Fig. 3. Mean plasma concentration time profile after intravenous administration of 10.0 mg/kg trifolirhizin in six rats.
spiked after extraction and multiplied by 100%. The recovery in plasma ranged from 78.5% to 86.4% for trifolirhizin. The recovery of IS (25 g/mL) in plasma was 87.4%. Assay performance data were presented in Table 1. The above results demonstrated that the values were within the acceptable range and the method was accurate and precise. 3.2.4. Stability Stability tests were performed at the low, medium and high QC samples with five determinations for each under different storage conditions. The RSDs of the mean test responses were within 15% in all stability tests. There was no statistically significant effect on the quantitation for plasma samples kept at room temperature for 24 h. No significant degradation was observed when samples of trifolirhizin were taken through three freeze (−20 ◦ C)–thaw (room temperature) cycles. As a result, trifolirhizin in samples were stable at −20 ◦ C for 31 days. 3.3. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral administration of 10.0 mg/kg trifolirhizin was shown in Fig. 3. The main Table 2 The main pharmacokinetic parameters after oral administration of 10.0 mg/kg trifolirhizin in six rats. Parameters
Trifolirhizin
t1/2 (h) Vd (L/kg) MRT (h) CL (L/h/kg) Cmax (ng/mL) AUC0 → t (ng/mL h) AUC0 → ∞ (ng/mL h)
0.68 16.65 0.65 17.14 1066.83 552.44 599.56
± ± ± ± ± ± ±
0.15 3.84 0.09 3.26 125.70 91.26 101.03
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