Journal of Chromatography B, 944 (2014) 6–10
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short communication
Determination of YZG-331 in mouse plasma using liquid chromatography–tandem mass spectrometry Zhihao Liu, Li Sheng, Yan Li ∗ Department of Drug Metabolism, Key Laboratory of Active Substances Discovery and Drug Ability Evaluation, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 1 September 2013 Accepted 28 October 2013 Available online 8 November 2013 Keywords: YZG-331 LC–MS/MS Mouse Pharmacokinetic
a b s t r a c t A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of YZG-331 in mouse plasma was developed. Plasma samples containing YZG-331 and YZG-441 (internal standard, IS) were prepared using a simple protein precipitation by the addition of acetonitrile. Thermo Scientific TSQ Quantum triple quadrupole system with multiple reaction monitoring (MRM) positive scanning mode was applied. The separation was performed on a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm) at a flow rate of 0.2 mL/min, using acetonitrile/water containing 0.1% formic acid (v/v) as mobile phase. The MS/MS ion transit ions monitored were 386→254 for YZG-331 and 400→268 for IS. Linear detection responses were obtained for YZG-331 ranging from 25 to 5000 ng/mL and the lower limits of quantitation (LLOQ) for the compound was 25 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 12.6% for all analytes, while the deviation of assay accuracies was within ±6.9%. The average recoveries of analytes were greater than 94.3%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of YZG-331 in mice. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Insomnia is an extremely common symptom and affects sleep quality and quantity, leading to the physical and mental health impairments [1,2]. Pharmacologic strategies have considered to be the major therapy to relieve insomnia in clinic. However, the existing sedative hypnotics show many unpleasant responses, such as drug tolerance, dependence and rebound insomnia [3,4]. To develop the new effective drugs with less adverse reactions for insomnia is still valuable. Previous studies have clearly shown that adenosine is an endogenous sleep factor via interacting with adenosine receptors [5,6]. Adenosine A1 and A2A receptors are believed to involve in the hypnogenic effect of adenosine in different areas of brain [7,8]. The rhizomes of Gastrodia elata have been used for the treatment of insomnia in oriental countries [9]. N6-(4-hydroxybenzyl) adenine riboside (NHBA) was found to be one of the bioactive components
∗ Corresponding author at: Department of Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing 100050, China. Tel.: +86 10 63165172; fax: +86 10 63165172. E-mail address: [email protected] (Y. Li). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.10.041
of G. elata [10,11], which played the important role on cell division and differentiation, anti-tumor and hypnotic activity [5,12]. YZG-331, a synthetic NHBA analog, exhibited the potent sedative and hypnotic effects by binding to adenosine A1 receptor in mice [13]. It is considered as a promising candidate for the treatment of insomnia at present. The objective of present study was to explore a simple, sensitive and accurate liquid chromatography with tandem mass spectrometry (LC–MS/MS) analytical method for the determination of bioavailability of YZG-331 in mice.
2. Experimental 2.1. Chemicals and reagent YZG-331 (purity > 99%) and YZG-441 (IS, purity > 99%) were synthesized at the Department of Natural Products Chemistry (Institute of Materia Medica, Chinese Academy of Medical Sciences) as described previously [13]. The chemical structures of YZG-331 and IS were identified by X-crystal diffraction, nuclear magnetic resonance, MS, infrared spectra, and elemental analysis. Acetonitrile was of HPLC grade (Fisher, USA). All other chemicals were of analytical reagent grade. Ultrapure water, prepared using a Milli-Q
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
Reagent water system (Millipore, MA, USA), was applied throughout the study. 2.2. Instruments The LC–MS/MS system consisted of a Surveyorauto-sampler, a Surveyor LC pump, a TSQ Quantum AccessTM triple quadrupole mass spectrometer with an electrospray ionization (ESI) source and Xcalibur 2.0a software for data acquisition and analysis (Thermo Finigan, USA). The analyte and IS were chromatographed by injection of 5 L sample onto a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm, Agilent, USA). The mobile phase consisted of solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1% formic acid in water). A gradient elution was applied by setting solvent A at 20% in 2 min and then increasing it at 95% in the next 3 min, followed by re-equilibration at 20% until 7.0 min. The flow rate was at 0.2 mL/min with an operating temperature at 30 ◦ C. The temperature of the auto-sampler was set at 4 ◦ C through out the analyses. An electrospray ionization (ESI) source was used in positive mode. The optimized ionspray voltage and temperature were set at 4000 V and 350 ◦ C, respectively. The sheath gas and auxiliary gas were nitrogen delivered at 30 psi and 10 L/min, respectively. The collision gas (argon) pressure and collision energy (CE) were 1.5 mTorr and 20 eV. Quantification was performed using the multiple reaction monitoring (MRM) transition m/z 386→254 for YZG-331 and m/z 400→268 for IS. 2.3. Preparation of standard solutions, plasma calibrators and QCs, and mouse plasma samples Standard stock solutions of YZG-331 and YZG-441 were dissolved in methanol at the concentration of 1 mg/mL. Working standards were prepared by dilution of the stock solution in acetonitrile to obtain the desired concentrations of 25, 50, 100, 200, 400, 1000, 2000, 5000 ng/mL for YZG-331. Plasma calibrators were prepared individually by mixing 10 L of IS working solution (1 g/mL), working standards, blank mouse plasma and 80 L of acetonitrile. The quality control (QC) samples containing 50, 500 and 2000 ng/mL of YZG-331 were prepared in a manner similar to that used for preparation of the calibrator samples. Mouse plasma samples of YZG-331 were prepared by mixing 10 L of plasma, IS working solution (1 g/mL) and 90 L of acetonitrile. The mixture was vortex mixed followed by centrifugation at 14,000 rpm for 5 min. A 5 L aliquot of each supernatant was injected into the LC–MS/MS system for the analysis. 2.4. Method validation 2.4.1. Selectivity The selectivity of the method was evaluated by analyzing the plasma samples collected from six mice to investigate the potential interferences at the peak regions for YZG-331 and IS. Chromatographic peaks of YZG-331 and IS were identified on the basis of their retention times and SRM responses. 2.4.2. Matrix effects The matrix effect was defined as the ion suppression/enhancement on the ionization of analytes, which was evaluated by comparing the responses of the deproteinized samples of blank plasma from six mice spiked QC samples with those of the standard solutions at equivalent concentrations. Experiments were performed at three QC concentration levels of YZG-331 in five replicates.
7
2.4.3. Linearity, precision and accuracy Plasma samples were quantified using the ratio of the peak area of analyte to IS as the assay response. The calibration curve required a correlation coefficient (r2 ) of 0.99 or better. The precision and accuracy of the assay were determined from QC samples. Intra-day assay precision and accuracy were estimated by analyzing five replicates on the same day at three QC levels, i.e., 50, 500 and 2000 ng/mL. Inter-assay precision was determined by analyzing the three levels of QC samples on five consecutive days. The variability of determination was expressed as the relative standard deviation (R.S.D.%) and the accuracy was expressed as the relative error (R.E.%). The criteria for acceptability of the data included an accuracy within ±15% R.E. from the nominal values and a precision of within ±15% R.S.D., except for LLOQ, where it should not exceed ±20% of accuracy as well as precision.
2.4.4. Stability studies Three freeze-thaws, long-term, short-term and postpreparative stabilities of YZG-331 in plasma were tested using QC samples. Freeze-thaw stability was evaluated in up to three cycles. In each freeze–thaw cycle, the samples were frozen and stored at −20 ◦ C for 24 h, then thawed at room temperature. To evaluate long-term stability of YZG-331, the plasma samples were stored at −20 ◦ C for 10 days. For the short-term stability, fresh plasma samples were kept at ambient temperature for 12 h before sample preparation. The stability of the prepared plasma samples was tested after keeping the treated samples at 4 ◦ C for 12 h. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% R.E.) and precision (±15% R.S.D.).
2.4.5. Recovery The recoveries of YZG-331 from mouse plasma were determined by comparing peak area ratios from regularly pretreated QC samples with those obtained from the direct injection of pure analyte standard solutions at three QC concentration levels. The recoveries of YZG-331 in mouse plasma were examined at least five times.
2.4.6. Pharmacokinetic study To assess the applicability of present method, the pharmacokinetic of YZG-331 was determined in mice after oral administration (n = 5). All animal protocols were approved by Institute Animal Care and Welfare Committee. ICR mice (adult male, 28 ± 2 g), were purchased from Beijing Vital River Experimental Animal Co., Ltd. The mice were allowed free access to water and chow diet and were fasted for 12 h with water ad libitum before pharmacokinetic experiments. The dosing solutions used for the experiments were prepared by suspending YZG-331 in 0.5% sodium carboxymethyl cellulose or 1% ethanol/physiological saline for oral and intravenous injection, respectively. The mice were given YZG-331 at dose of 10 mg/kg or 1 mg/kg via the gavages and tail vein injection. Then 50 L of blood samples were collected via the orbital plexus into heparinized tubes at 5, 15, 30 min, 1, 2, 4, 6, 8 h after oral dosing and 2, 5, 10, 20, 30 min, 1, 2, 4, 6 h after i.v. administration. Plasma (10 L) was immediately separated by centrifugation at 5000 rpm for 10 min. All samples were stored at −20 ◦ C until analyzed. The pharmacokinetic parameters of YZG-331 were calculated using WinNonlin software version 6.3 based on noncompartmental analysis (Pharsight Corporation, Mountain View, USA). The parameters considered were: Cmax (maximum plasma concentration), AUC (area under the concentration–time curve), T1/2 (plasma half-life in the terminal phase), CLp (plasma clearance) and the bioavailability (F = AUCp.o. /AUCi.v × 100).
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Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
Fig. 1. Product ion mass spectra of [M+H]+ of (A) YZG-331 and (B) YZG-441.
3. Results and discussion
mouse plasma, LLOQ plasma sample and an in vivo plasma sample at 30 min after oral administration (Fig. 2).
3.1. Method development Different ionization methods (including positive and negative modes) were compared to obtain better specificity and sensitivity for analyte determination. The positive ionization mode was found to be more appropriate for YZG-331 detection than the negative mode. The positive ion electrospray mass spectra of YZG-331 and IS in full-scan Q1 mode showed a protonated molecular ion [M+H]+ as the base peak, m/z 386 for YZG-331 and m/z 400 for IS. As shown in Fig. 1, the most stable and abundant productions were at m/z 254 for YZG-331 and 268 for IS, after fragmentation in the collision cell. The most suitable CEs for the analytes were determined by observing the response of the obtained fragment ion peaks. To get appropriate retention time, better resolution and sensitivity, the different HPLC parameters including category of column, mobile phase and flow rate of mobile phase were tested and compared. The mobile phase of acetonitrile/water containing 0.1% formic acid (v/v) at a flow of 0.2 mL/min was found to be suitable for the determination of electrospray responses of analytes. During method development, a number of reversed-phase C18 columns, such as Zorbax SB-C18, Hypersil BDS C18, and Gemini were tested. At last, a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm) was adopted to achieve symmetric and sharp peak shapes for both YZG331 and IS.
3.2. Method validation 3.2.1. Selectivity Under the described LC–MS/MS conditions, the retention time of YZG-331 and IS were 4.1 and 4.2 min, respectively, while no interference peak was detected at related retention times in blank
3.2.2. Matrix effect The matrix effect values were 100.51–106.04% for YZG-331 at different QC levels, respectively. The R.S.D. values of the matrix effect values were 3.3–6.3%, indicating that no significant matrix effect was observed in any of the plasma sample under our experimental conditions. 3.2.3. Calibration curve A liner relationship was found between peak area ratios and YZG-331 concentrations within the range of 25–5000 ng/mL. The typical equation of the calibration curves was as follows: (r2 = 0.9994). Y = 0.031065 + 0.00221789*X − 3.85243e−008*X∧ 2 The LLOQ of YZG-331 was 25 ng/mL in mouse plasma. 3.2.4. Precision and accuracy The precision and accuracy of the method for YZG-331 were assessed by determining QC samples (n = 5) at three concentrations and summarized in Table 1. The intra- and inter-day precisions were below 8.8% and 12.6%, respectively, with relative errors from −4.6% to 6.9%. The results indicate that the present LC–MS/MS method was accurate, reliable and reproducible. 3.2.5. Recovery and stability The recoveries of YZG-331 at three QC concentration levels (n = 5) in plasma were 94.34–103.00%. All stability results are shown in Table 2. The samples were found to be stable after being placed at room temperature for 12 h, stored at −20 ◦ C for 10 days or through freeze–thaw cycles in mouse plasma. Further more, samples after treatment were stable at 4 ◦ C in auto-sampler for 12 h, which indicated that a large number of samples could be determined in each analytical run.
Table 1 Intra- and inter-day accuracies and precisions of YZG-331 in mouse plasma (n = 5). Nominal concentration (ng/mL)
Measured (mean ± S.D.)
Accuracy (R.E.%)
Intra-day (n = 5)
50 500 2000
53.44 ± 4.73 511.35 ± 13.43 1973.02 ± 32.02.09
6.9 2.3 −1.3
8.8 2.6 1.6
Intra-day (n = 5)
50 500 2000
51.00 ± 6.42 483.58 ± 44.59 1908.54 ± 139.86
2.0 −3.3 −4.6
12.6 9.2 7.3
Percision (R.S.D.%)
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
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Fig. 2. Typical SRM chromatograms of YZG-331 and IS: (A) blank mouse plasma; (B) LLOQ plasma sample; and (C) mouse plasma sample at 30 min post dose of YZG-331 (10 mg/kg) spiked with IS. (a) YZG-331 (m/z 386–254); (b) IS (m/z 400–268).
Table 2 Stability of YZG-331 in mouse plasma (n = 5). Conditions
Nominal concentration (ng/mL)
Measured (mean ± S.D.)
Accuracy (R.E.%)
Room temperature (24 h)
50 500 2000
54.78 ± 1.98 498.78 ± 18.16 1968.65 ± 58.09
9.6 −0.2 −1.6
One freeze–thaw cycles
50 500 2000
54.73 ± 3.28 504.78 ± 14.61 2080.43 ± 56.81
9.5 1.0 4.0
Three freeze–thaw cycles
50 500 2000
55.22 ± 3.00 507.92 ± 24.41 2083.83 ± 69.66
10.4 1.6 4.2
At 4 ◦ C (12 h)
50 500 2000
52.52 ± 2.26 518.08 ± 10.72 2038.08 ± 130.13
Stored at −20 ◦ C (10days)
50 500 2000
49.60 ± 3.77 486.78 ± 21.70 1981.78 ± 41.97
5.0 3.6 1.9 −0.8 −2.6 −0.9
10
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
Fig. 3. Mean plasma concentration curves of YZG-331 in mice after p.o. (10 mg/kg) or intravenous (1 mg/kg).
Table 3 Pharmacokinetic parameters of YZG-331 after oral or intravenous administration. Values represent means ± S.D. (n = 5). Parameters
Unit
Oral
Intravenous
t1/2z Tmax Cmax AUC(0−t) CLz
h h g/L g/L h mL/h/kg
4.1 ± 0.3 0.3 3282.3 ± 658.3 4300.6 ± 966.2 2377.8 ± 489.5
4.2 ± – 732.8 ± 757.6 ± 1302.3 ±
2.9 37.5 189.8 338.9
Acknowledgments This work was supported by the National Science and Technology Major Project of China (2012ZX09301002-001-007, 2011ZX09102-001-01, 2012ZX09301002-006, 2012ZX09103-101001) as well as PUMC Youth Fund and Fundamental Research Funds for the Central Universities (3332013073). The authors would like to acknowledge Ms. Hu, Ms. Chen and Mr. Wang for their technical assistance. References
3.3. Pharmacokinetic study The method was successfully employed to determine YZG-331 concentrations in mouse plasma samples. The mean plasma concentration and major pharmacokinetic parameters were shown in Fig. 3 and Table 3. A comparison between AUCs of YZG-331 obtained after oral and intravenous treatment, revealed good absorption with the bioavailability of 56.8%. 4. Conclusion In conclusion, the newly developed LC–MS/MS method with simple protein precipitation provided a sensitive, reproducible and validated assay for the determination of YZG-331 in mouse plasma. The method was successfully applied to the pharmacokinetic studies of YZG-331 in mice.
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N.L. Borja, K.L. Daniel, Clin. Ther. 28 (2006) 1540. K. Ramar, E.J. Olson, Am. Fam. Physician 88 (2013) 231. A.N. Griffiths, D.M. Jones, A. Richens, Br. J. Clin. Pharmacol. 21 (1986) 647. M.L. Bocca, F. Le Doze, O. Etard, M. Pottier, J. L’Hoste, P. Denise, Psychopharmacology (Berl.) 143 (1999) 373. Y. Zhang, M. Li, R.X. Kang, J.G. Shi, G.T. Liu, J.J. Zhang, Pharmacol. Biochem. Behav. 102 (2012) 450. T.E. Scammell, D.Y. Gerashchenko, T. Mochizuki, M.T. McCarthy, I.V. Estabrooke, C.A. Sears, C.B. Saper, Y. Urade, O. Hayaishi, Neuroscience 107 (2001) 653. O. Hayaishi, Y. Urade, N. Eguchi, Z.L. Huang, Arch. Ital. Biol. 142 (2004) 533. Z.L. Huang, Y. Urade, O. Hayaishi, Curr. Top. Med. Chem. 11 (2011) 1047. C.F. Tsai, C.L. Huang, Y.L. Lin, Y.C. Lee, Y.C. Yang, N.K. Huang, J. Ethnopharmacol. 138 (2011) 119. N.K. Huang, Y. Chern, J.M. Fang, C.I. Lin, W.P. Chen, Y.L. Lin, J. Nat. Prod. 70 (2007) 571. Y. Lei, L. Wang, M. Cheng, H. Xiao, Biomed. Chromatogr. 25 (2011) 344. N.K. Huang, J.H. Lin, J.T. Lin, C.I. Lin, E.M. Liu, C.J. Lin, W.P. Chen, Y.C. Shen, H.M. Chen, J.B. Chen, H.L. Lai, C.W. Yang, M.C. Chiang, Y.S. Wu, C. Chang, J.F. Chen, J.M. Fang, Y.L. Lin, Y. Chern, PLoS ONE 6 (2011) e20934. J. Shi, Z. Yue, M. Li, C. Zhu, WO2011069294 (2011/06/26).
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Short communication
Determination of YZG-331 in mouse plasma using liquid chromatography–tandem mass spectrometry Zhihao Liu, Li Sheng, Yan Li ∗ Department of Drug Metabolism, Key Laboratory of Active Substances Discovery and Drug Ability Evaluation, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e
i n f o
Article history: Received 1 September 2013 Accepted 28 October 2013 Available online 8 November 2013 Keywords: YZG-331 LC–MS/MS Mouse Pharmacokinetic
a b s t r a c t A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of YZG-331 in mouse plasma was developed. Plasma samples containing YZG-331 and YZG-441 (internal standard, IS) were prepared using a simple protein precipitation by the addition of acetonitrile. Thermo Scientific TSQ Quantum triple quadrupole system with multiple reaction monitoring (MRM) positive scanning mode was applied. The separation was performed on a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm) at a flow rate of 0.2 mL/min, using acetonitrile/water containing 0.1% formic acid (v/v) as mobile phase. The MS/MS ion transit ions monitored were 386→254 for YZG-331 and 400→268 for IS. Linear detection responses were obtained for YZG-331 ranging from 25 to 5000 ng/mL and the lower limits of quantitation (LLOQ) for the compound was 25 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 12.6% for all analytes, while the deviation of assay accuracies was within ±6.9%. The average recoveries of analytes were greater than 94.3%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of YZG-331 in mice. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Insomnia is an extremely common symptom and affects sleep quality and quantity, leading to the physical and mental health impairments [1,2]. Pharmacologic strategies have considered to be the major therapy to relieve insomnia in clinic. However, the existing sedative hypnotics show many unpleasant responses, such as drug tolerance, dependence and rebound insomnia [3,4]. To develop the new effective drugs with less adverse reactions for insomnia is still valuable. Previous studies have clearly shown that adenosine is an endogenous sleep factor via interacting with adenosine receptors [5,6]. Adenosine A1 and A2A receptors are believed to involve in the hypnogenic effect of adenosine in different areas of brain [7,8]. The rhizomes of Gastrodia elata have been used for the treatment of insomnia in oriental countries [9]. N6-(4-hydroxybenzyl) adenine riboside (NHBA) was found to be one of the bioactive components
∗ Corresponding author at: Department of Metabolism, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing 100050, China. Tel.: +86 10 63165172; fax: +86 10 63165172. E-mail address: [email protected] (Y. Li). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.10.041
of G. elata [10,11], which played the important role on cell division and differentiation, anti-tumor and hypnotic activity [5,12]. YZG-331, a synthetic NHBA analog, exhibited the potent sedative and hypnotic effects by binding to adenosine A1 receptor in mice [13]. It is considered as a promising candidate for the treatment of insomnia at present. The objective of present study was to explore a simple, sensitive and accurate liquid chromatography with tandem mass spectrometry (LC–MS/MS) analytical method for the determination of bioavailability of YZG-331 in mice.
2. Experimental 2.1. Chemicals and reagent YZG-331 (purity > 99%) and YZG-441 (IS, purity > 99%) were synthesized at the Department of Natural Products Chemistry (Institute of Materia Medica, Chinese Academy of Medical Sciences) as described previously [13]. The chemical structures of YZG-331 and IS were identified by X-crystal diffraction, nuclear magnetic resonance, MS, infrared spectra, and elemental analysis. Acetonitrile was of HPLC grade (Fisher, USA). All other chemicals were of analytical reagent grade. Ultrapure water, prepared using a Milli-Q
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
Reagent water system (Millipore, MA, USA), was applied throughout the study. 2.2. Instruments The LC–MS/MS system consisted of a Surveyorauto-sampler, a Surveyor LC pump, a TSQ Quantum AccessTM triple quadrupole mass spectrometer with an electrospray ionization (ESI) source and Xcalibur 2.0a software for data acquisition and analysis (Thermo Finigan, USA). The analyte and IS were chromatographed by injection of 5 L sample onto a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm, Agilent, USA). The mobile phase consisted of solvent A (0.1% formic acid in acetonitrile) and solvent B (0.1% formic acid in water). A gradient elution was applied by setting solvent A at 20% in 2 min and then increasing it at 95% in the next 3 min, followed by re-equilibration at 20% until 7.0 min. The flow rate was at 0.2 mL/min with an operating temperature at 30 ◦ C. The temperature of the auto-sampler was set at 4 ◦ C through out the analyses. An electrospray ionization (ESI) source was used in positive mode. The optimized ionspray voltage and temperature were set at 4000 V and 350 ◦ C, respectively. The sheath gas and auxiliary gas were nitrogen delivered at 30 psi and 10 L/min, respectively. The collision gas (argon) pressure and collision energy (CE) were 1.5 mTorr and 20 eV. Quantification was performed using the multiple reaction monitoring (MRM) transition m/z 386→254 for YZG-331 and m/z 400→268 for IS. 2.3. Preparation of standard solutions, plasma calibrators and QCs, and mouse plasma samples Standard stock solutions of YZG-331 and YZG-441 were dissolved in methanol at the concentration of 1 mg/mL. Working standards were prepared by dilution of the stock solution in acetonitrile to obtain the desired concentrations of 25, 50, 100, 200, 400, 1000, 2000, 5000 ng/mL for YZG-331. Plasma calibrators were prepared individually by mixing 10 L of IS working solution (1 g/mL), working standards, blank mouse plasma and 80 L of acetonitrile. The quality control (QC) samples containing 50, 500 and 2000 ng/mL of YZG-331 were prepared in a manner similar to that used for preparation of the calibrator samples. Mouse plasma samples of YZG-331 were prepared by mixing 10 L of plasma, IS working solution (1 g/mL) and 90 L of acetonitrile. The mixture was vortex mixed followed by centrifugation at 14,000 rpm for 5 min. A 5 L aliquot of each supernatant was injected into the LC–MS/MS system for the analysis. 2.4. Method validation 2.4.1. Selectivity The selectivity of the method was evaluated by analyzing the plasma samples collected from six mice to investigate the potential interferences at the peak regions for YZG-331 and IS. Chromatographic peaks of YZG-331 and IS were identified on the basis of their retention times and SRM responses. 2.4.2. Matrix effects The matrix effect was defined as the ion suppression/enhancement on the ionization of analytes, which was evaluated by comparing the responses of the deproteinized samples of blank plasma from six mice spiked QC samples with those of the standard solutions at equivalent concentrations. Experiments were performed at three QC concentration levels of YZG-331 in five replicates.
7
2.4.3. Linearity, precision and accuracy Plasma samples were quantified using the ratio of the peak area of analyte to IS as the assay response. The calibration curve required a correlation coefficient (r2 ) of 0.99 or better. The precision and accuracy of the assay were determined from QC samples. Intra-day assay precision and accuracy were estimated by analyzing five replicates on the same day at three QC levels, i.e., 50, 500 and 2000 ng/mL. Inter-assay precision was determined by analyzing the three levels of QC samples on five consecutive days. The variability of determination was expressed as the relative standard deviation (R.S.D.%) and the accuracy was expressed as the relative error (R.E.%). The criteria for acceptability of the data included an accuracy within ±15% R.E. from the nominal values and a precision of within ±15% R.S.D., except for LLOQ, where it should not exceed ±20% of accuracy as well as precision.
2.4.4. Stability studies Three freeze-thaws, long-term, short-term and postpreparative stabilities of YZG-331 in plasma were tested using QC samples. Freeze-thaw stability was evaluated in up to three cycles. In each freeze–thaw cycle, the samples were frozen and stored at −20 ◦ C for 24 h, then thawed at room temperature. To evaluate long-term stability of YZG-331, the plasma samples were stored at −20 ◦ C for 10 days. For the short-term stability, fresh plasma samples were kept at ambient temperature for 12 h before sample preparation. The stability of the prepared plasma samples was tested after keeping the treated samples at 4 ◦ C for 12 h. Samples were considered stable if assay values were within the acceptable limits of accuracy (±15% R.E.) and precision (±15% R.S.D.).
2.4.5. Recovery The recoveries of YZG-331 from mouse plasma were determined by comparing peak area ratios from regularly pretreated QC samples with those obtained from the direct injection of pure analyte standard solutions at three QC concentration levels. The recoveries of YZG-331 in mouse plasma were examined at least five times.
2.4.6. Pharmacokinetic study To assess the applicability of present method, the pharmacokinetic of YZG-331 was determined in mice after oral administration (n = 5). All animal protocols were approved by Institute Animal Care and Welfare Committee. ICR mice (adult male, 28 ± 2 g), were purchased from Beijing Vital River Experimental Animal Co., Ltd. The mice were allowed free access to water and chow diet and were fasted for 12 h with water ad libitum before pharmacokinetic experiments. The dosing solutions used for the experiments were prepared by suspending YZG-331 in 0.5% sodium carboxymethyl cellulose or 1% ethanol/physiological saline for oral and intravenous injection, respectively. The mice were given YZG-331 at dose of 10 mg/kg or 1 mg/kg via the gavages and tail vein injection. Then 50 L of blood samples were collected via the orbital plexus into heparinized tubes at 5, 15, 30 min, 1, 2, 4, 6, 8 h after oral dosing and 2, 5, 10, 20, 30 min, 1, 2, 4, 6 h after i.v. administration. Plasma (10 L) was immediately separated by centrifugation at 5000 rpm for 10 min. All samples were stored at −20 ◦ C until analyzed. The pharmacokinetic parameters of YZG-331 were calculated using WinNonlin software version 6.3 based on noncompartmental analysis (Pharsight Corporation, Mountain View, USA). The parameters considered were: Cmax (maximum plasma concentration), AUC (area under the concentration–time curve), T1/2 (plasma half-life in the terminal phase), CLp (plasma clearance) and the bioavailability (F = AUCp.o. /AUCi.v × 100).
8
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
Fig. 1. Product ion mass spectra of [M+H]+ of (A) YZG-331 and (B) YZG-441.
3. Results and discussion
mouse plasma, LLOQ plasma sample and an in vivo plasma sample at 30 min after oral administration (Fig. 2).
3.1. Method development Different ionization methods (including positive and negative modes) were compared to obtain better specificity and sensitivity for analyte determination. The positive ionization mode was found to be more appropriate for YZG-331 detection than the negative mode. The positive ion electrospray mass spectra of YZG-331 and IS in full-scan Q1 mode showed a protonated molecular ion [M+H]+ as the base peak, m/z 386 for YZG-331 and m/z 400 for IS. As shown in Fig. 1, the most stable and abundant productions were at m/z 254 for YZG-331 and 268 for IS, after fragmentation in the collision cell. The most suitable CEs for the analytes were determined by observing the response of the obtained fragment ion peaks. To get appropriate retention time, better resolution and sensitivity, the different HPLC parameters including category of column, mobile phase and flow rate of mobile phase were tested and compared. The mobile phase of acetonitrile/water containing 0.1% formic acid (v/v) at a flow of 0.2 mL/min was found to be suitable for the determination of electrospray responses of analytes. During method development, a number of reversed-phase C18 columns, such as Zorbax SB-C18, Hypersil BDS C18, and Gemini were tested. At last, a ZorbaxSB-C18 column (3.5 m, 2.1 mm × 100 mm) was adopted to achieve symmetric and sharp peak shapes for both YZG331 and IS.
3.2. Method validation 3.2.1. Selectivity Under the described LC–MS/MS conditions, the retention time of YZG-331 and IS were 4.1 and 4.2 min, respectively, while no interference peak was detected at related retention times in blank
3.2.2. Matrix effect The matrix effect values were 100.51–106.04% for YZG-331 at different QC levels, respectively. The R.S.D. values of the matrix effect values were 3.3–6.3%, indicating that no significant matrix effect was observed in any of the plasma sample under our experimental conditions. 3.2.3. Calibration curve A liner relationship was found between peak area ratios and YZG-331 concentrations within the range of 25–5000 ng/mL. The typical equation of the calibration curves was as follows: (r2 = 0.9994). Y = 0.031065 + 0.00221789*X − 3.85243e−008*X∧ 2 The LLOQ of YZG-331 was 25 ng/mL in mouse plasma. 3.2.4. Precision and accuracy The precision and accuracy of the method for YZG-331 were assessed by determining QC samples (n = 5) at three concentrations and summarized in Table 1. The intra- and inter-day precisions were below 8.8% and 12.6%, respectively, with relative errors from −4.6% to 6.9%. The results indicate that the present LC–MS/MS method was accurate, reliable and reproducible. 3.2.5. Recovery and stability The recoveries of YZG-331 at three QC concentration levels (n = 5) in plasma were 94.34–103.00%. All stability results are shown in Table 2. The samples were found to be stable after being placed at room temperature for 12 h, stored at −20 ◦ C for 10 days or through freeze–thaw cycles in mouse plasma. Further more, samples after treatment were stable at 4 ◦ C in auto-sampler for 12 h, which indicated that a large number of samples could be determined in each analytical run.
Table 1 Intra- and inter-day accuracies and precisions of YZG-331 in mouse plasma (n = 5). Nominal concentration (ng/mL)
Measured (mean ± S.D.)
Accuracy (R.E.%)
Intra-day (n = 5)
50 500 2000
53.44 ± 4.73 511.35 ± 13.43 1973.02 ± 32.02.09
6.9 2.3 −1.3
8.8 2.6 1.6
Intra-day (n = 5)
50 500 2000
51.00 ± 6.42 483.58 ± 44.59 1908.54 ± 139.86
2.0 −3.3 −4.6
12.6 9.2 7.3
Percision (R.S.D.%)
Z. Liu et al. / J. Chromatogr. B 944 (2014) 6–10
9
Fig. 2. Typical SRM chromatograms of YZG-331 and IS: (A) blank mouse plasma; (B) LLOQ plasma sample; and (C) mouse plasma sample at 30 min post dose of YZG-331 (10 mg/kg) spiked with IS. (a) YZG-331 (m/z 386–254); (b) IS (m/z 400–268).
Table 2 Stability of YZG-331 in mouse plasma (n = 5). Conditions
Nominal concentration (ng/mL)
Measured (mean ± S.D.)
Accuracy (R.E.%)
Room temperature (24 h)
50 500 2000
54.78 ± 1.98 498.78 ± 18.16 1968.65 ± 58.09
9.6 −0.2 −1.6
One freeze–thaw cycles
50 500 2000
54.73 ± 3.28 504.78 ± 14.61 2080.43 ± 56.81
9.5 1.0 4.0
Three freeze–thaw cycles
50 500 2000
55.22 ± 3.00 507.92 ± 24.41 2083.83 ± 69.66
10.4 1.6 4.2
At 4 ◦ C (12 h)
50 500 2000
52.52 ± 2.26 518.08 ± 10.72 2038.08 ± 130.13
Stored at −20 ◦ C (10days)
50 500 2000
49.60 ± 3.77 486.78 ± 21.70 1981.78 ± 41.97
5.0 3.6 1.9 −0.8 −2.6 −0.9
10
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Fig. 3. Mean plasma concentration curves of YZG-331 in mice after p.o. (10 mg/kg) or intravenous (1 mg/kg).
Table 3 Pharmacokinetic parameters of YZG-331 after oral or intravenous administration. Values represent means ± S.D. (n = 5). Parameters
Unit
Oral
Intravenous
t1/2z Tmax Cmax AUC(0−t) CLz
h h g/L g/L h mL/h/kg
4.1 ± 0.3 0.3 3282.3 ± 658.3 4300.6 ± 966.2 2377.8 ± 489.5
4.2 ± – 732.8 ± 757.6 ± 1302.3 ±
2.9 37.5 189.8 338.9
Acknowledgments This work was supported by the National Science and Technology Major Project of China (2012ZX09301002-001-007, 2011ZX09102-001-01, 2012ZX09301002-006, 2012ZX09103-101001) as well as PUMC Youth Fund and Fundamental Research Funds for the Central Universities (3332013073). The authors would like to acknowledge Ms. Hu, Ms. Chen and Mr. Wang for their technical assistance. References
3.3. Pharmacokinetic study The method was successfully employed to determine YZG-331 concentrations in mouse plasma samples. The mean plasma concentration and major pharmacokinetic parameters were shown in Fig. 3 and Table 3. A comparison between AUCs of YZG-331 obtained after oral and intravenous treatment, revealed good absorption with the bioavailability of 56.8%. 4. Conclusion In conclusion, the newly developed LC–MS/MS method with simple protein precipitation provided a sensitive, reproducible and validated assay for the determination of YZG-331 in mouse plasma. The method was successfully applied to the pharmacokinetic studies of YZG-331 in mice.
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