Journal of Chromatography B, 986–987 (2015) 18–22
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of acacetin in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study Li-hua Fan a,1 , Xiaoheng Li b,1 , De-yuan Chen a , Ning Zhang a , Yiyan Wang b , Yuanyuan Shan b , Yuanyuan Hu b , Ren-ai Xu b , Jian Jin c,∗∗ , Ren-Shan Ge b,∗ a
Department of Anesthesiology, the Sixth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China The 2nd Affiliated Hospital & Research Academy of Reproductive Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China c Department of Pharmacy, Shanghai No.9 People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China b
a r t i c l e
i n f o
Article history: Received 17 November 2014 Received in revised form 26 January 2015 Accepted 29 January 2015 Available online 7 February 2015 Keywords: Acacetin UPLC-MS/MS Rat plasma Pharmacokinetics
a b s t r a c t A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of acacetin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of acacetin was at 0.83 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 285.3 → 242.2 for acacetin and m/z 237.2 → 194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1–1600 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of acacetin in plasma was in the range of 78.4–85.2%. Intra-day and inter-day precision were both 98%) was purchased from Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Carbamazepine (internal standard, IS, purity > 98%) was purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was obtained using a Millipore 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 program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 50–95% B (0–0.9 min), 95–50% B (0.9–1.0 min). A subsequent re-equilibration time (1 min) was performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 5 L. The column and sample temperature were maintained at 30 ◦ C and 4 ◦ C, respectively. An AB Sciex QTRAP 5500 triple quadruple 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 285.3 → 242.2 and m/z 237.2 → 194.3 for acacetin and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 55 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV; and temperature, 500 ◦ C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex). 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of acacetin 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 method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the literatures for validation of bioanalytical methods (US Food and Drug Administration, 2001). 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. The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma spiked with acacetin and IS and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of acacetin to IS were plotted against analyte concentrations, and standard curves were fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 1–1600 ng/mL. The lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves; acacetin peak should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80–120%. To evaluate the matrix effect (ME), blank rat plasma were extracted and then spiked with the analyte at 2, 200 and 1200 ng/mL. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the ME. The ME of IS was evaluated at the working concentration (200 ng/mL) in a similar way. Precision and accuracy were assessed by the determination of QC samples at three concentration levels (2, 200 and 1200 ng/mL) in six replicates in three validation days. The precision was expressed by coefficient of variation (CV). The precision determined at each concentration level should not exceed 15% of the CV. Precision is subdivided into intra- and inter-day precision, which assesses precision during a single day run and between-day run, which measures precision with a day, and within three days. The recovery of acacetin was evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in the same manner. The stabilities of acacetin in rat plasma were evaluated by analyzing five replicates of plasma samples at the concentrations of 2, 200 and 1200 ng/mL, which were exposed to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 4 h, and the ready-to-inject samples (after protein precipitation) in the UPLC autosampler at room temperature for 24 h. The
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freeze-thaw stability was evaluated after three complete freezethaw 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 35 days. 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 acacetin. 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 College 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, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 h after intravenous administration of acacetin (5.0 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 acacetin concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China). 3. Results and discussion 3.1. Method development and optimization This method was intended for rapid quantification of the plasma concentrations after acacetin exposure. A simple one step protein precipitation procedure was chosen to shorten the sample preparation time. The optimization of the procedure was obtained after testing several precipitating solvents and different solvent compositions, such as acetonitrile, acetonitrile-methanol (9:1, v/v), and trichloroacetic acid-methanol (1:9, v/v). The results with the best recoveries and lowest ME for the analytes were achieved with the solvent of acetonitrile. High speed centrifugation at 13,000 × g for 8 min helped to remove tiny particles and increase the service life of the column. A number of commercially available UPLC columns and various mobile phases were evaluated for its chromatographic behavior and the ionization response of acacetin. Various combinations of organic diluents (methanol/acetonitrile) together with ammonium formate or formic acid were tested. The best response was obtained from acetonitrile and water (containing 0.1% formic acid). The addition of 0.1% formic acid to the mobile phase increased the sensitivity of acacetin. The gradient elution mode was applied in chromatographic separation and showed a better peak shape and appropriate retention time. Compared to Shim-pack XR-ODS III column (2.0 mm × 75 mm, 1.6 m), a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with isocratic delivery provided satisfactory chromatographic results with minimal ME. In this assay, no significant signal suppression or enhancement was found using the current conditions. The whole separation of the analyte and IS was completed within 1.0 min per sample. Acacetin and IS were eluted at about 0.83 and 0.52 min, respectively. 3.2. Selectivity and ME No interfering endogenous substance was observed at the retention time of the analyte and IS (Fig. 2). The ME for acacetin at concentrations of 2, 200 and 1200 ng/mL was measured to be 96.8 ± 4.9, 102.6 ± 5.3 and 103.5 ± 5.1% (n = 6), respectively. The ME for IS (200 ng/mL) was 99.3 ± 3.9% (n = 6). As a result, ME from plasma was negligible in this method.
Fig. 2. Representative chromatograms of acacetin and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with acacetin (100 ng/mL) and IS (200 ng/mL); (C) a rat plasma sample 30 min after intravenous administration of single dosage 5.0 mg/kg acacetin (IS: 200 ng/mL).
3.3. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 1–1600 ng/mL for acacetin in rat plasma. A typical equation of the calibration curve is: y = (0.0606 ± 0.0002)x + (0.1245 ± 0.0051), r = 0.9902, where y represents the ratios of acacetin peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of acacetin in plasma was 1 ng/mL. The precision and accuracy at LLOQ were 9.5 and 90.7%, respectively. The limit of detection, defined as a signal/noise ratio of 3, was 0.5 ng/mL for acacetin in plasma. 3.4. Precision, accuracy and recovery The precision of the method was determined by calculating CV for QCs at three concentration levels over three validation days. Intra-day precision was 9.4% or less and the inter-day precision was 10.5% or less at each QC level. The accuracy of the method ranged from 92.6 to 106.2% at each QC level. Mean recoveries of acacetin were better than 78.4%. The recovery of the IS (200 ng/mL) was 87.5 ± 5.8%. Assay performance data is presented in Table 1. The results demonstrate that the values are within the acceptable range and the method is accurate and precise.
L.-h. Fan et al. / J. Chromatogr. B 986–987 (2015) 18–22
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Table 1 Precision, accuracy and recovery for acacetin of quality control sample in rat plasma (n = 6). Analyte
Acacetin
Concentration (ng/mL)
CV (%)
1 2 200 1200
Recovery (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
10.3 9.4 7.4 2.6
11.2 10.5 9.1 3.9
95.4 92.6 105.2 97.6
98.3 106.4 93.3 98.8
78.4 85.2 81.4 82.1
± ± ± ±
3.8 5.1 4.7 4.7
Table 2 Summary of stability of acacetin under various storage conditions (n = 5). Condition
Concentration (ng/mL) Nominal
Found
CV (%)
Accuracy (%)
Ambient, 4 h
2 200 1200
2.1 ± 0.2 219.5 ± 14.1 1114.2 ± 99.7
9.2 6.4 9.0
103.1 109.8 92.9
Autosampler, ambient, 24 h
2 200 1200
1.8 ± 0.1 213.8 ± 11.7 1093.2 ± 82.8
8.1 5.5 7.6
90.2 106.9 91.1
Three freeze-thaw
2 200 1200
1.9 ± 0.2 193.2 ± 15.5 1096.1 ± 88.8
9.2 8.0 8.1
94.7 96.6 91.3
−20 ◦ C, 35 days
2 200 1200
2.0 ± 0.1 186.3 ± 11.4 1118.4 ± 75.4
7.3 6.1 6.7
101.4 93.2 93.2
3.5. Stability The autosampler, room temperature, freeze-thaw and longterm (35 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of nominal values, and the established method was suitable for the pharmacokinetic study (Table 2). 3.6. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The plasma samples with analyte concentrations above the upper limit of quantitation were diluted with blank rat plasma. The mean plasma concentration–time curve after intravenous administration of 5.0 mg/kg acacetin was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 3. After intravenous administration of acacetin, a mean maximum plasma concentration (Cmax ) was found to be 1334.9 ± 211.6 ng/mL.
Table 3 The main pharmacokinetic parameters after intravenous administration of 5.0 mg/kg acacetin in ten rats. Parameters
Acacetin
t1/2 (h) Cmax (ng/mL) AUC0→t (ng/mL h) AUC0→∞ (ng/mL h) MRT0→t (h) MRT0→∞ (h)
1.48 1334.9 1111.8 1120.5 0.95 1.02
± ± ± ± ± ±
0.53 211.6 140.7 146.6 0.06 0.11
The plasma concentration of acacetin decreased rapidly and was eliminated from plasma with a terminal half-life of 1.48 ± 0.53 h. The initial rapid decline in the plasma concentration indicates that the compound might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings. 4. Conclusions A UPLC-MS/MS method for the determination of acacetin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of acacetin level in rat plasma using an UPLC-MS/MS method. The method offered sample preparation with a simple one-step precipitation of plasma protein by acetonitrile 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 acacetin in rats. References
Fig. 3. Mean plasma concentration time profile after intravenous administration of 5.0 mg/kg acacetin in six rats.
[1] L. Marzocchella, M. Fantini, M. Benvenuto, L. Masuelli, I. Tresoldi, A. Modesti, R. Bei, Recent Patents Inflamm. Allergy Drug Discov. 5 (2011) 200. [2] K.H. Shen, S.H. Hung, L.T. Yin, C.S. Huang, C.H. Chao, C.L. Liu, Y.W. Shih, Mol. Cell. Biochem. 333 (2010) 279. [3] S.T. Chien, S.S. Lin, C.K. Wang, Y.B. Lee, K.S. Chen, Y. Fong, Y.W. Shih, Mol. Cell. Biochem. 350 (2011) 135. [4] K. Watanabe, S. Kanno, A. Tomizawa, S. Yomogida, M. Ishikawa, Oncol. Rep. 27 (2012) 204.
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[5] T.A. Bhat, D. Nambiar, D. Tailor, A. Pal, R. Agarwal, R.P. Singh, Cancer Prev. Res. 6 (2013) 1128. [6] N. Tanigawa, M. Hagiwara, H. Tada, T. Komatsu, S. Sugiura, K. Kobayashi, Y. Kato, N. Ishida, K. Nishida, M. Ninomiya, M. Koketsu, K. Matsushita, Immunopharmacol. Immunotoxicol. 35 (2013) 471. [7] J.H. Bian, K. Qian, X. Xu, J. Shen, Zhong yao cai=Zhongyaocai=J. Chin. Med. Mater. 29 (2006) 1233. [8] H. Shen, Q. Guo, H. Fang, Y. Wang, M. Jin, Zhongguo Zhong yao za zhi=Zhongguo zhongyao zazhi=Chin. J. Chin. Mater. Med. 35 (2010) 191. [9] S.E. Nielsen, L.O. Dragsted, J. Chromatogr. B, Biomed. Sci. Appl (1998) 379.
[10] A. Nugroho, S.C. Lim, J.S. Byeon, J.S. Choi, H.J. Park, J. Pharm. Biomed. Analysis 76 (2013) 139. [11] S.F. Wang, J. Leng, Y.M. Xu, M.L. Feng, J. Zhejiang Univ. Sci. B 14 (2013) 604. [12] X.L. Wu, C.W. Li, H.M. Chen, Z.Q. Su, X.N. Zhao, J.N. Chen, X.P. Lai, X.J. Zhang, Z.R. Su, Evid. Complement. Alternat. Med.: eCAM 2013 (2013) 413237. [13] A. Vijaya Bhaskar Reddy, N. Venugopal, G. Madhavi, K. Gangadhara Reddy, V. Madhavi, J. Pharm. Biomed. Analysis 84 (2013) 84. [14] W. Xiong, X. Tao, S. Pang, X. Yang, G. Tang, Z. Bian, J. Chromatogr. Sci. (2013). [15] D. Agnesod, A. De Nicolo, M. Simiele, A. Mohamed Abdi, L. Boglione, G. Di Perri, A. D’Avolio, J. Pharm. Biomed. Analysis 90C (2013) 119.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of acacetin in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study Li-hua Fan a,1 , Xiaoheng Li b,1 , De-yuan Chen a , Ning Zhang a , Yiyan Wang b , Yuanyuan Shan b , Yuanyuan Hu b , Ren-ai Xu b , Jian Jin c,∗∗ , Ren-Shan Ge b,∗ a
Department of Anesthesiology, the Sixth Affiliated Hospital of Wenzhou Medical University, Lishui 323000, Zhejiang, China The 2nd Affiliated Hospital & Research Academy of Reproductive Biomedicine, Wenzhou Medical University, Wenzhou, Zhejiang 325027, China c Department of Pharmacy, Shanghai No.9 People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China b
a r t i c l e
i n f o
Article history: Received 17 November 2014 Received in revised form 26 January 2015 Accepted 29 January 2015 Available online 7 February 2015 Keywords: Acacetin UPLC-MS/MS Rat plasma Pharmacokinetics
a b s t r a c t A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of acacetin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of acacetin was at 0.83 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 285.3 → 242.2 for acacetin and m/z 237.2 → 194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1–1600 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of acacetin in plasma was in the range of 78.4–85.2%. Intra-day and inter-day precision were both 98%) was purchased from Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Carbamazepine (internal standard, IS, purity > 98%) was purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were of HPLC grade and were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was obtained using a Millipore 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 program was employed with the mobile phase combining solvent A (0.1% formic acid in water) and solvent B (acetonitrile) as follows: 50–95% B (0–0.9 min), 95–50% B (0.9–1.0 min). A subsequent re-equilibration time (1 min) was performed before next injection. The flow rate was 0.40 mL/min and the injection volume was 5 L. The column and sample temperature were maintained at 30 ◦ C and 4 ◦ C, respectively. An AB Sciex QTRAP 5500 triple quadruple 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 285.3 → 242.2 and m/z 237.2 → 194.3 for acacetin and IS, respectively. After optimization, the source parameters were set as follows: curtain gas, 35 psig; nebulizer gas, 55 psig; turbo gas, 60 psig; ion spray voltage, 4.0 kV; and temperature, 500 ◦ C. Data acquiring and processing were performed using analyst software (version 1.5, AB Sciex). 2.3. Standard solutions, calibration standards and quality control (QC) sample The stock solution of acacetin 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 method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the literatures for validation of bioanalytical methods (US Food and Drug Administration, 2001). 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. The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma spiked with acacetin and IS and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of acacetin to IS were plotted against analyte concentrations, and standard curves were fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 1–1600 ng/mL. The lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves; acacetin peak should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80–120%. To evaluate the matrix effect (ME), blank rat plasma were extracted and then spiked with the analyte at 2, 200 and 1200 ng/mL. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations, and this peak area ratio is defined as the ME. The ME of IS was evaluated at the working concentration (200 ng/mL) in a similar way. Precision and accuracy were assessed by the determination of QC samples at three concentration levels (2, 200 and 1200 ng/mL) in six replicates in three validation days. The precision was expressed by coefficient of variation (CV). The precision determined at each concentration level should not exceed 15% of the CV. Precision is subdivided into intra- and inter-day precision, which assesses precision during a single day run and between-day run, which measures precision with a day, and within three days. The recovery of acacetin was evaluated by comparing peak area ratios of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in the same manner. The stabilities of acacetin in rat plasma were evaluated by analyzing five replicates of plasma samples at the concentrations of 2, 200 and 1200 ng/mL, which were exposed to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 4 h, and the ready-to-inject samples (after protein precipitation) in the UPLC autosampler at room temperature for 24 h. The
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L.-h. Fan et al. / J. Chromatogr. B 986–987 (2015) 18–22
freeze-thaw stability was evaluated after three complete freezethaw 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 35 days. 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 acacetin. 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 College 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, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 h after intravenous administration of acacetin (5.0 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 acacetin concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Shanghai University of Traditional Chinese Medicine, China). 3. Results and discussion 3.1. Method development and optimization This method was intended for rapid quantification of the plasma concentrations after acacetin exposure. A simple one step protein precipitation procedure was chosen to shorten the sample preparation time. The optimization of the procedure was obtained after testing several precipitating solvents and different solvent compositions, such as acetonitrile, acetonitrile-methanol (9:1, v/v), and trichloroacetic acid-methanol (1:9, v/v). The results with the best recoveries and lowest ME for the analytes were achieved with the solvent of acetonitrile. High speed centrifugation at 13,000 × g for 8 min helped to remove tiny particles and increase the service life of the column. A number of commercially available UPLC columns and various mobile phases were evaluated for its chromatographic behavior and the ionization response of acacetin. Various combinations of organic diluents (methanol/acetonitrile) together with ammonium formate or formic acid were tested. The best response was obtained from acetonitrile and water (containing 0.1% formic acid). The addition of 0.1% formic acid to the mobile phase increased the sensitivity of acacetin. The gradient elution mode was applied in chromatographic separation and showed a better peak shape and appropriate retention time. Compared to Shim-pack XR-ODS III column (2.0 mm × 75 mm, 1.6 m), a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 m) with isocratic delivery provided satisfactory chromatographic results with minimal ME. In this assay, no significant signal suppression or enhancement was found using the current conditions. The whole separation of the analyte and IS was completed within 1.0 min per sample. Acacetin and IS were eluted at about 0.83 and 0.52 min, respectively. 3.2. Selectivity and ME No interfering endogenous substance was observed at the retention time of the analyte and IS (Fig. 2). The ME for acacetin at concentrations of 2, 200 and 1200 ng/mL was measured to be 96.8 ± 4.9, 102.6 ± 5.3 and 103.5 ± 5.1% (n = 6), respectively. The ME for IS (200 ng/mL) was 99.3 ± 3.9% (n = 6). As a result, ME from plasma was negligible in this method.
Fig. 2. Representative chromatograms of acacetin and IS in rat plasma samples. (A) a blank plasma sample; (B) a blank plasma sample spiked with acacetin (100 ng/mL) and IS (200 ng/mL); (C) a rat plasma sample 30 min after intravenous administration of single dosage 5.0 mg/kg acacetin (IS: 200 ng/mL).
3.3. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 1–1600 ng/mL for acacetin in rat plasma. A typical equation of the calibration curve is: y = (0.0606 ± 0.0002)x + (0.1245 ± 0.0051), r = 0.9902, where y represents the ratios of acacetin peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of acacetin in plasma was 1 ng/mL. The precision and accuracy at LLOQ were 9.5 and 90.7%, respectively. The limit of detection, defined as a signal/noise ratio of 3, was 0.5 ng/mL for acacetin in plasma. 3.4. Precision, accuracy and recovery The precision of the method was determined by calculating CV for QCs at three concentration levels over three validation days. Intra-day precision was 9.4% or less and the inter-day precision was 10.5% or less at each QC level. The accuracy of the method ranged from 92.6 to 106.2% at each QC level. Mean recoveries of acacetin were better than 78.4%. The recovery of the IS (200 ng/mL) was 87.5 ± 5.8%. Assay performance data is presented in Table 1. The results demonstrate that the values are within the acceptable range and the method is accurate and precise.
L.-h. Fan et al. / J. Chromatogr. B 986–987 (2015) 18–22
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Table 1 Precision, accuracy and recovery for acacetin of quality control sample in rat plasma (n = 6). Analyte
Acacetin
Concentration (ng/mL)
CV (%)
1 2 200 1200
Recovery (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
10.3 9.4 7.4 2.6
11.2 10.5 9.1 3.9
95.4 92.6 105.2 97.6
98.3 106.4 93.3 98.8
78.4 85.2 81.4 82.1
± ± ± ±
3.8 5.1 4.7 4.7
Table 2 Summary of stability of acacetin under various storage conditions (n = 5). Condition
Concentration (ng/mL) Nominal
Found
CV (%)
Accuracy (%)
Ambient, 4 h
2 200 1200
2.1 ± 0.2 219.5 ± 14.1 1114.2 ± 99.7
9.2 6.4 9.0
103.1 109.8 92.9
Autosampler, ambient, 24 h
2 200 1200
1.8 ± 0.1 213.8 ± 11.7 1093.2 ± 82.8
8.1 5.5 7.6
90.2 106.9 91.1
Three freeze-thaw
2 200 1200
1.9 ± 0.2 193.2 ± 15.5 1096.1 ± 88.8
9.2 8.0 8.1
94.7 96.6 91.3
−20 ◦ C, 35 days
2 200 1200
2.0 ± 0.1 186.3 ± 11.4 1118.4 ± 75.4
7.3 6.1 6.7
101.4 93.2 93.2
3.5. Stability The autosampler, room temperature, freeze-thaw and longterm (35 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of nominal values, and the established method was suitable for the pharmacokinetic study (Table 2). 3.6. Application of the method in a pharmacokinetic study The method was applied to a pharmacokinetic study in rats. The plasma samples with analyte concentrations above the upper limit of quantitation were diluted with blank rat plasma. The mean plasma concentration–time curve after intravenous administration of 5.0 mg/kg acacetin was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 3. After intravenous administration of acacetin, a mean maximum plasma concentration (Cmax ) was found to be 1334.9 ± 211.6 ng/mL.
Table 3 The main pharmacokinetic parameters after intravenous administration of 5.0 mg/kg acacetin in ten rats. Parameters
Acacetin
t1/2 (h) Cmax (ng/mL) AUC0→t (ng/mL h) AUC0→∞ (ng/mL h) MRT0→t (h) MRT0→∞ (h)
1.48 1334.9 1111.8 1120.5 0.95 1.02
± ± ± ± ± ±
0.53 211.6 140.7 146.6 0.06 0.11
The plasma concentration of acacetin decreased rapidly and was eliminated from plasma with a terminal half-life of 1.48 ± 0.53 h. The initial rapid decline in the plasma concentration indicates that the compound might have left the plasma and been distributed into the other tissues, but further studies will be conducted to confirm these findings. 4. Conclusions A UPLC-MS/MS method for the determination of acacetin in rat plasma was developed and validated. To the best of our knowledge, this is the first report of the determination of acacetin level in rat plasma using an UPLC-MS/MS method. The method offered sample preparation with a simple one-step precipitation of plasma protein by acetonitrile 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 acacetin in rats. References
Fig. 3. Mean plasma concentration time profile after intravenous administration of 5.0 mg/kg acacetin in six rats.
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