Journal of Chromatography B, 944 (2014) 35–38
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of sec-O-glucosylhamaudol in rat plasma by gradient elution liquid chromatography–mass spectrometry Congcong Wen a , Chongliang Lin b , Xiaojun Cai c , Jianshe Ma c , Xianqin Wang c,∗ a b c
Laboratory Animal Centre of Wenzhou Medical University, Wenzhou 325035, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China Analytical and Testing Center of Wenzhou Medical University, Wenzhou 325035, China
a r t i c l e
i n f o
Article history: Received 7 September 2013 Accepted 1 November 2013 Available online 15 November 2013 Keywords: Sec-O-glucosylhamaudol LC–MS Pharmacokinetics Rat plasma
a b s t r a c t Sec-O-glucosylhamaudol is one of the major bioactive compounds of the Saposhnikoviae Radix. A simple and selective liquid chromatography–mass spectrometry (LC–MS) method for determination of sec-O-glucosylhamaudol in rat plasma was developed. After addition of carbamazepine as internal standard (IS), protein precipitation with acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 m) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 439 for sec-O-glucosylhamaudol and m/z 237 for the IS. Calibration plots were linear over the range of 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma. Mean recovery of sec-O-glucosylhamaudol in plasma was in the range of 74.8–83.7%. Intra-day and inter-day precision were both 98%) was purchased from the Chengdu Mansite Pharmaceutical Company Limited. (Chengdu, China). Carbamazepine (IS, purity > 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA). All other chemicals were analytical grade and used without further purification. 2.2. Instrumentation and conditions
∗ Corresponding author at: Analytical and Testing Center, Wenzhou Medical University, University-Town, Wenzhou 325035, China. Tel.: +86 57786699156; fax: +86 57786699156. E-mail address: [email protected] (X. Wang). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.11.001
Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany)
36
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
2.4. Sample preparation Before sample treatment, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (2.0 g/mL) was added to 100 L of collected plasma sample followed by the addition of 200 L acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min, the supernatant (2 L) was injected into the LC–ESI-MS for analysis. 2.5. Method validation
Fig. 1. Chemical structure of sec-O-glucosylhamaudol (a) and carbamazepine (IS, b).
controlled by ChemStation (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1 mm × 150 mm, 5 m) column at 30 ◦ C, with acetonitrile–0.1% formic acid as mobile phase. The flow rate was 0.4 mL/min. A gradient elution program was conducted for chromatographic separation with mobile phase A (0.1% formic acid), and mobile phase B (acetonitrile) as follows: 0–4.0 min (10–80% B), 4.0–7.0 min (80–80% B), 7.0–8.0 min (80–10% B), 8.0–13.0 min (10–10% B). Drying gas flow and nebulizer pressure were set at 6 L/min and 25 psi, respectively. Dry gas temperature and capillary voltage of the system were adjusted at 350 ◦ C and 3500 V, respectively. LC-MS was performed with SIM mode using target ions at m/z 439 for sec-O-glucosylhamaudol and m/z 237 for carbamazepine (IS), in positive ion ESI interface, respectively.
2.3. Calibration standards and quality control samples The stock solution of sec-O-glucosylhamaudol (100 g/mL) and carbamazepine (IS) (100 g/mL) were prepared in methanol–water (50: 50), repectively. Working solutions for calibration and controls were prepared from the stock solution by dilution with methanol. The 2.0 g/mL working standard solution of IS was prepared by dilution of the IS stock solution with methanol. All of the solutions were stored at 4 ◦ C and were brought to room temperature before use. Sec-O-glucosylhamaudol calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma (50, 100, 200, 500, 1000, 2000, 4000 and 8000 ng/mL). Quality-control (QC) samples (100, 1000 and 6000 ng/mL) were prepared by the same way as the calibration standards. The analytical standards and QC samples were stored at −20 ◦ C.
Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of sec-O-glucosylhamaudol to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 50–8000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves; sec-O-glucosylhamaudol peak should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80–120%. Precision and accuracy were assessed by the determination of QC samples 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 sec-O-glucosylhamaudol 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 a similar way. The selectivity of the method was evaluated by analyzing blank plasma spiked sec-O-glucosylhamaudol and IS. To evaluate the matrix effect, blank rat plasma was extracted and then spiked with the analyte at 100, 1000 and 6000 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 matrix effect (ME). The ME of IS was evaluated at the working concentration (200 ng/mL) in the same manner. Carry-over was assessed following injection of a blank plasma sample immediately after 3 repeats of the upper limit of quantification (ULOQ) and the response was checked [10]. The stabilities of sec-O-glucosylhamaudol in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 100 and 6000 ng/mL, which were exposed to different conditions [11]. 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 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature 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 20 days. The stability of the IS (200 ng/mL) was evaluated in a similar way. 2.6. Pharmacokinetic study Male Sprague–Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of sec-Oglucosylhamaudol. All six rats were housed at Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
37
Fig. 2. Representative LC–MS chromatograms of sec-O-glucosylhamaudol (1) and carbamazepine (IS, 2), (a) blank plasma; (b) blank plasma spiked with sec-Oglucosylhamaudol (50 ng/mL) and IS (200 ng/mL); (c) a rat plasma sample 3 h after intravenous administration of single dosage 2.5 mg/kg sec-O-glucosylhamaudol.
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, 0.0833, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 h after intravenous administration of secO-glucosylhamaudol (2.5 mg/kg). The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma sec-O-glucosylhamaudol concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
accuracy at LLOQ were 17.8% and 116.3%, respectively. The LOD of sec-O-glucosylhamaudol in rat plasma was 20 ng/mL, defined as a signal/noise ratio of 3.
3.2. Precision, accuracy and recovery Intra-day precision was 15% or less and the inter-day precision was 14% or less at each QC level. The accuracy of the method ranged from 93.6% to 103.1% at each QC level. Assay performance data are presented in Table 1. Mean recoveries of sec-O-glucosylhamaudol at concentrations of 100, 1000 and 6000 ng/mL were measured to be 74.8–83.7%. The recovery of the IS (200 ng/mL) was 94.6 ± 3.5%.
3. Results and discussion 3.3. Selectivity and matrix effect 3.1. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma. Typical equation of the calibration curve was: y = 0.000133 x − 0.004617, r = 0.99555, where y represents the ratios of sec-O-glucosylhamaudol peak area to that of IS and x represents the plasma concentration. The LLOQ for secO-glucosylhamaudol in plasma was 50 ng/mL. The precision and
Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with sec-O-glucosylhamaudol and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The ME for sec-O-glucosylhamaudol at concentrations of 100, 1000 and 6000 ng/mL was measured to be 86.9–94.0% (Table 1). The ME for IS (200 ng/mL) was measured to 96.7 ± 4.2% (n = 6). As a result, ME from plasma was negligible in this method.
Table 1 Precision and accuracy for sec-O-glucosylhamaudol of QC sample in rat plasma (n = 6). Concentration (ng/mL)
100 1000 6000
CV (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
14.2 7.3 7.2
13.6 12.9 7.7
93.6 100.0 103.1
95.6 101.3 99.1
Matrix effects
Extraction recovery
90.2 ± 6.8 94.0 ± 8.2 86.9 ± 4.3
83.7 ± 9.0 74.8 ± 6.4 77.0 ± 4.2
38
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
3.5. Stability The results of auto-sampler, room temperature, freeze–thaw and long-term (20 days) stability indicated that the analyte was stable under the various storage conditions since the bias in concentration was within ±15% of their nominal values. 3.6. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 2. 4. Conclusion
Fig. 3. Mean plasma concentration time profile after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol in six rats. Table 2 The main pharmacokinetic parameters after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol in six rats. Parameters
Unit
Mean (± SD)
AUC(0–t) AUC(0–∞) MRT(0–t) MRT(0–∞) t1/2 CL V Cmax
ng/mL × h ng/mL × h h h h L/h/kg L/kg ng/mL
4921.0 ± 913.2 4929.4 ± 910.1 1.49 ± 0.12 1.52 ± 0.14 1.29 ± 0.32 0.5 ± 0.1 1.0 ± 0.3 5608.9 ± 1442.6
3.4. Carry-over None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding 5 extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over [10].
The LC-MS method described here allowed accurate and reproducible method for determination of sec-O-glucosylhamaudol in rat plasma utilizing 100 L of plasma with an LLOQ of 50 ng/mL. The LC-MS method successfully applied to a pharmacokinetic study after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol to rats. References [1] Z.G. Zheng, R.S. Wang, H.Q. Cheng, T.T. Duan, B. He, D. Tang, F. Gu, Q. Zhu, J. Pharm. Biomed. Anal. 54 (2011) 614–618. [2] J. Kang, L. Zhou, J.H. Sun, M. Ye, J. Han, B.R. Wang, D.A. Guo, J. Asian Nat. Prod. Res. 10 (2008) 971–976. [3] E. Okuyama, T. Hasegawa, T. Matsushita, H. Fujimoto, M. Ishibashi, M. Yamazaki, Chem. Pharm. Bull. 49 (2001) 154–160. [4] R. Liu, S. Wu, A. Sun, Phytochem. Anal.: PCA 19 (2008) 206–211. [5] W. Li, Z. Wang, Y.S. Sun, L. Chen, L.K. Han, Y.N. Zheng, Phytochem. Anal.: PCA 22 (2011) 313–321. [6] M.K. Kim, D.H. Yang, M. Jung, E.H. Jung, H.Y. Eom, J.H. Suh, J.W. Min, U. Kim, H. Min, J. Kim, S.B. Han, J. Chromatogr. A 1218 (2011) 6319–6330. [7] Y. Gui, R. Tsao, L. Li, C.M. Liu, J. Wang, X. Zong, J. Sep. Sci. 34 (2011) 520–526. [8] Y.Q. Xiao, B. Yang, S.T. Yao, W. Li, L. Li, L.Q. Huang, B.Y. Xue, Zhongguo Zhong yao za zhi=Zhongguo zhongyao zazhi 26 (2001) 185–187. [9] W.W. You, Z.H. Wu, M. Zou, X.M. Tan, Nan fang yi ke da xue xue bao 27 (2007) 884–886. [10] S. Ghassabian, S.M. Moosavi, Y.G. Valero, K. Shekar, J.F. Fraser, M.T. Smith, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 903 (2012) 126–133. [11] K. Liu, D.F. Zhong, X.Y. Chen, J. Chromatogr. B 878 (2010) 2415–2420.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short Communication
Determination of sec-O-glucosylhamaudol in rat plasma by gradient elution liquid chromatography–mass spectrometry Congcong Wen a , Chongliang Lin b , Xiaojun Cai c , Jianshe Ma c , Xianqin Wang c,∗ a b c
Laboratory Animal Centre of Wenzhou Medical University, Wenzhou 325035, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China Analytical and Testing Center of Wenzhou Medical University, Wenzhou 325035, China
a r t i c l e
i n f o
Article history: Received 7 September 2013 Accepted 1 November 2013 Available online 15 November 2013 Keywords: Sec-O-glucosylhamaudol LC–MS Pharmacokinetics Rat plasma
a b s t r a c t Sec-O-glucosylhamaudol is one of the major bioactive compounds of the Saposhnikoviae Radix. A simple and selective liquid chromatography–mass spectrometry (LC–MS) method for determination of sec-O-glucosylhamaudol in rat plasma was developed. After addition of carbamazepine as internal standard (IS), protein precipitation with acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 m) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 439 for sec-O-glucosylhamaudol and m/z 237 for the IS. Calibration plots were linear over the range of 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma. Mean recovery of sec-O-glucosylhamaudol in plasma was in the range of 74.8–83.7%. Intra-day and inter-day precision were both 98%) was purchased from the Chengdu Mansite Pharmaceutical Company Limited. (Chengdu, China). Carbamazepine (IS, purity > 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA, USA). All other chemicals were analytical grade and used without further purification. 2.2. Instrumentation and conditions
∗ Corresponding author at: Analytical and Testing Center, Wenzhou Medical University, University-Town, Wenzhou 325035, China. Tel.: +86 57786699156; fax: +86 57786699156. E-mail address: [email protected] (X. Wang). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.11.001
Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany)
36
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
2.4. Sample preparation Before sample treatment, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 L of the IS working solution (2.0 g/mL) was added to 100 L of collected plasma sample followed by the addition of 200 L acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 × g for 10 min, the supernatant (2 L) was injected into the LC–ESI-MS for analysis. 2.5. Method validation
Fig. 1. Chemical structure of sec-O-glucosylhamaudol (a) and carbamazepine (IS, b).
controlled by ChemStation (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1 mm × 150 mm, 5 m) column at 30 ◦ C, with acetonitrile–0.1% formic acid as mobile phase. The flow rate was 0.4 mL/min. A gradient elution program was conducted for chromatographic separation with mobile phase A (0.1% formic acid), and mobile phase B (acetonitrile) as follows: 0–4.0 min (10–80% B), 4.0–7.0 min (80–80% B), 7.0–8.0 min (80–10% B), 8.0–13.0 min (10–10% B). Drying gas flow and nebulizer pressure were set at 6 L/min and 25 psi, respectively. Dry gas temperature and capillary voltage of the system were adjusted at 350 ◦ C and 3500 V, respectively. LC-MS was performed with SIM mode using target ions at m/z 439 for sec-O-glucosylhamaudol and m/z 237 for carbamazepine (IS), in positive ion ESI interface, respectively.
2.3. Calibration standards and quality control samples The stock solution of sec-O-glucosylhamaudol (100 g/mL) and carbamazepine (IS) (100 g/mL) were prepared in methanol–water (50: 50), repectively. Working solutions for calibration and controls were prepared from the stock solution by dilution with methanol. The 2.0 g/mL working standard solution of IS was prepared by dilution of the IS stock solution with methanol. All of the solutions were stored at 4 ◦ C and were brought to room temperature before use. Sec-O-glucosylhamaudol calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma (50, 100, 200, 500, 1000, 2000, 4000 and 8000 ng/mL). Quality-control (QC) samples (100, 1000 and 6000 ng/mL) were prepared by the same way as the calibration standards. The analytical standards and QC samples were stored at −20 ◦ C.
Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of sec-O-glucosylhamaudol to IS were plotted against analyte concentrations, and standard curves were well fitted to the equations by linear regression with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 50–8000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves; sec-O-glucosylhamaudol peak should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80–120%. Precision and accuracy were assessed by the determination of QC samples 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 sec-O-glucosylhamaudol 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 a similar way. The selectivity of the method was evaluated by analyzing blank plasma spiked sec-O-glucosylhamaudol and IS. To evaluate the matrix effect, blank rat plasma was extracted and then spiked with the analyte at 100, 1000 and 6000 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 matrix effect (ME). The ME of IS was evaluated at the working concentration (200 ng/mL) in the same manner. Carry-over was assessed following injection of a blank plasma sample immediately after 3 repeats of the upper limit of quantification (ULOQ) and the response was checked [10]. The stabilities of sec-O-glucosylhamaudol in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 100 and 6000 ng/mL, which were exposed to different conditions [11]. 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 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature 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 20 days. The stability of the IS (200 ng/mL) was evaluated in a similar way. 2.6. Pharmacokinetic study Male Sprague–Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of sec-Oglucosylhamaudol. All six rats were housed at Wenzhou Medical University Laboratory Animal Research Center. All experimental procedures and protocols were reviewed and approved by the
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
37
Fig. 2. Representative LC–MS chromatograms of sec-O-glucosylhamaudol (1) and carbamazepine (IS, 2), (a) blank plasma; (b) blank plasma spiked with sec-Oglucosylhamaudol (50 ng/mL) and IS (200 ng/mL); (c) a rat plasma sample 3 h after intravenous administration of single dosage 2.5 mg/kg sec-O-glucosylhamaudol.
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, 0.0833, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8 h after intravenous administration of secO-glucosylhamaudol (2.5 mg/kg). The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 L) was stored at −20 ◦ C until analysis. Plasma sec-O-glucosylhamaudol concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
accuracy at LLOQ were 17.8% and 116.3%, respectively. The LOD of sec-O-glucosylhamaudol in rat plasma was 20 ng/mL, defined as a signal/noise ratio of 3.
3.2. Precision, accuracy and recovery Intra-day precision was 15% or less and the inter-day precision was 14% or less at each QC level. The accuracy of the method ranged from 93.6% to 103.1% at each QC level. Assay performance data are presented in Table 1. Mean recoveries of sec-O-glucosylhamaudol at concentrations of 100, 1000 and 6000 ng/mL were measured to be 74.8–83.7%. The recovery of the IS (200 ng/mL) was 94.6 ± 3.5%.
3. Results and discussion 3.3. Selectivity and matrix effect 3.1. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 50–8000 ng/mL for sec-O-glucosylhamaudol in rat plasma. Typical equation of the calibration curve was: y = 0.000133 x − 0.004617, r = 0.99555, where y represents the ratios of sec-O-glucosylhamaudol peak area to that of IS and x represents the plasma concentration. The LLOQ for secO-glucosylhamaudol in plasma was 50 ng/mL. The precision and
Fig. 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with sec-O-glucosylhamaudol and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The ME for sec-O-glucosylhamaudol at concentrations of 100, 1000 and 6000 ng/mL was measured to be 86.9–94.0% (Table 1). The ME for IS (200 ng/mL) was measured to 96.7 ± 4.2% (n = 6). As a result, ME from plasma was negligible in this method.
Table 1 Precision and accuracy for sec-O-glucosylhamaudol of QC sample in rat plasma (n = 6). Concentration (ng/mL)
100 1000 6000
CV (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
14.2 7.3 7.2
13.6 12.9 7.7
93.6 100.0 103.1
95.6 101.3 99.1
Matrix effects
Extraction recovery
90.2 ± 6.8 94.0 ± 8.2 86.9 ± 4.3
83.7 ± 9.0 74.8 ± 6.4 77.0 ± 4.2
38
C. Wen et al. / J. Chromatogr. B 944 (2014) 35–38
3.5. Stability The results of auto-sampler, room temperature, freeze–thaw and long-term (20 days) stability indicated that the analyte was stable under the various storage conditions since the bias in concentration was within ±15% of their nominal values. 3.6. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol was shown in Fig. 3. The main pharmacokinetic parameters from non-compartment model analysis were summarized in Table 2. 4. Conclusion
Fig. 3. Mean plasma concentration time profile after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol in six rats. Table 2 The main pharmacokinetic parameters after intravenous administration of 2.5 mg/kg sec-O-glucosylhamaudol in six rats. Parameters
Unit
Mean (± SD)
AUC(0–t) AUC(0–∞) MRT(0–t) MRT(0–∞) t1/2 CL V Cmax
ng/mL × h ng/mL × h h h h L/h/kg L/kg ng/mL
4921.0 ± 913.2 4929.4 ± 910.1 1.49 ± 0.12 1.52 ± 0.14 1.29 ± 0.32 0.5 ± 0.1 1.0 ± 0.3 5608.9 ± 1442.6
3.4. Carry-over None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding 5 extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over [10].
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