Journal of Chromatography B, 963 (2014) 24–28
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation a liquid chromatography mass spectrometry for determination of solasodine in rat plasma and its application to a pharmacokinetic study Jianshe Ma a , Xitao Ding a , Chengxiang Sun b , Chongliang Lin b , Xinxin An b , Guanyang Lin b , Xuezhi Yang b , Xianqin Wang c,∗ a
Function Experiment Teaching Center of Wenzhou Medical University, Wenzhou 325035, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c Analytical and Testing Center of Wenzhou Medical University, University-town, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 7 February 2014 Accepted 15 May 2014 Available online 23 May 2014 Keywords: Solasodine LC–MS Pharmacokinetics Rat plasma
a b s t r a c t Solasodine is a poisonous alkaloid chemical compound that occurs in plants of the Solanaceae family. A simple and selective liquid chromatography mass spectrometry method for determination of solasodine in rat plasma was developed and validated over the range of 3–1000 ng/mL. Chromatographic separation was achieved on a C18 (2.1 mm × 50 mm, 3.5 m) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. After addition of midazolam as internal standard (IS), liquid–liquid extraction by ethyl acetate was used as sample preparation. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification with target ions m/z 414 for solasodine and m/z 326 for IS. Mean recoveries of solasodine in rat plasma were in the range of 87.6–94.1%. Matrix effects for solasodine were between 94.9% and 102.3%. Coefficient of variation of intra-day and inter-day precision were both 98%) was purchased from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Midazolam (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 Millipore Milli-Q purification system (Bedford, MA, USA).
J. Ma et al. / J. Chromatogr. B 963 (2014) 24–28
25
Fig. 1. Chemical structure of solasodine (a) and midazolam (IS, b).
2.2. Instrumentation and conditions
2.4. Sample preparation
Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) controlled by ChemStation software (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on an Agilent Zorbax SB–C18 (2.1 mm × 50 mm, 3.5 m) column at 30 ◦ C, with acetonitrile–0.1% formic acid as mobile phase. The flow rate was set at 0.4 mL/min. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water), and mobile phase B (acetonitrile) as follows: 0–2.0 min (10–80% B), 2.0–5.0 min (80% B), 5.0–6.0 min (80–10% B), 6.0–10.0 min (10% B). Drying gas flow and nebuliser pressure were set at 6 L/min and 25 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 ◦ C and 4000 V, respectively. The smart target was set at m/z 400. LC–MS was performed with selective ion monitoring mode using target ions at m/z 414 for solasodine (Fig. 2a) and m/z 326 for midazolam (IS, Fig. 2b), in positive ion electrospray ionization interface. The ion at m/z 414 is the protonated molecule [M + H]+ for solasodine, and the ion at m/z 326 is the protonated molecule [M + H]+ for IS.
Before analysis, 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 1.0 mL ethyl acetate. The tubes were vortex mixed for 1.0 min. After centrifugation at 5000 × g for 10 min, the supernatant organic layer was transferred into a 2 mL glass tube and dried under nitrogen stream at 40 ◦ C. The dried residue was reconstituted in 150 L of methanol–water (50:50, v/v) and a 5 L aliquot of this was injected into LC–MS. 2.5. Method validation The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set
2.3. Calibration standards and quality control samples The stock solutions of solasodine (1.0 mg/mL) and midazolam (IS, 1.0 mg/mL) were prepared in methanol, respectively. 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 brought to room temperature before use. Solasodine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 3–1000 ng/mL for solasodine in rat plasma, prepared at concentrations of 3, 6, 10, 20, 50, 100, 200, 500 and 1000 ng/mL. Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations (8, 420 and 840 ng/mL). The calibration standards and QC samples were stored at −20 ◦ C.
Fig. 2. Mass spectrum of solasodine (a) and midazolam (IS, b).
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J. Ma et al. / J. Chromatogr. B 963 (2014) 24–28
by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) [28,29]. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC samples. The selectivity of the method was evaluated by analyzing six blank rat plasma, blank plasma spiked solasodine and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of solasodine 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 3–1000 ng/mL. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curves, which can be quantified reliably, with an acceptable accuracy (80–120%) and precision (
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Development and validation a liquid chromatography mass spectrometry for determination of solasodine in rat plasma and its application to a pharmacokinetic study Jianshe Ma a , Xitao Ding a , Chengxiang Sun b , Chongliang Lin b , Xinxin An b , Guanyang Lin b , Xuezhi Yang b , Xianqin Wang c,∗ a
Function Experiment Teaching Center of Wenzhou Medical University, Wenzhou 325035, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c Analytical and Testing Center of Wenzhou Medical University, University-town, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 7 February 2014 Accepted 15 May 2014 Available online 23 May 2014 Keywords: Solasodine LC–MS Pharmacokinetics Rat plasma
a b s t r a c t Solasodine is a poisonous alkaloid chemical compound that occurs in plants of the Solanaceae family. A simple and selective liquid chromatography mass spectrometry method for determination of solasodine in rat plasma was developed and validated over the range of 3–1000 ng/mL. Chromatographic separation was achieved on a C18 (2.1 mm × 50 mm, 3.5 m) column with acetonitrile–0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. After addition of midazolam as internal standard (IS), liquid–liquid extraction by ethyl acetate was used as sample preparation. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification with target ions m/z 414 for solasodine and m/z 326 for IS. Mean recoveries of solasodine in rat plasma were in the range of 87.6–94.1%. Matrix effects for solasodine were between 94.9% and 102.3%. Coefficient of variation of intra-day and inter-day precision were both 98%) was purchased from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Midazolam (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 Millipore Milli-Q purification system (Bedford, MA, USA).
J. Ma et al. / J. Chromatogr. B 963 (2014) 24–28
25
Fig. 1. Chemical structure of solasodine (a) and midazolam (IS, b).
2.2. Instrumentation and conditions
2.4. Sample preparation
Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) controlled by ChemStation software (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany). Chromatographic separation was achieved on an Agilent Zorbax SB–C18 (2.1 mm × 50 mm, 3.5 m) column at 30 ◦ C, with acetonitrile–0.1% formic acid as mobile phase. The flow rate was set at 0.4 mL/min. A gradient elution programme was conducted for chromatographic separation with mobile phase A (0.1% formic acid in water), and mobile phase B (acetonitrile) as follows: 0–2.0 min (10–80% B), 2.0–5.0 min (80% B), 5.0–6.0 min (80–10% B), 6.0–10.0 min (10% B). Drying gas flow and nebuliser pressure were set at 6 L/min and 25 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 ◦ C and 4000 V, respectively. The smart target was set at m/z 400. LC–MS was performed with selective ion monitoring mode using target ions at m/z 414 for solasodine (Fig. 2a) and m/z 326 for midazolam (IS, Fig. 2b), in positive ion electrospray ionization interface. The ion at m/z 414 is the protonated molecule [M + H]+ for solasodine, and the ion at m/z 326 is the protonated molecule [M + H]+ for IS.
Before analysis, 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 1.0 mL ethyl acetate. The tubes were vortex mixed for 1.0 min. After centrifugation at 5000 × g for 10 min, the supernatant organic layer was transferred into a 2 mL glass tube and dried under nitrogen stream at 40 ◦ C. The dried residue was reconstituted in 150 L of methanol–water (50:50, v/v) and a 5 L aliquot of this was injected into LC–MS. 2.5. Method validation The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set
2.3. Calibration standards and quality control samples The stock solutions of solasodine (1.0 mg/mL) and midazolam (IS, 1.0 mg/mL) were prepared in methanol, respectively. 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 brought to room temperature before use. Solasodine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 3–1000 ng/mL for solasodine in rat plasma, prepared at concentrations of 3, 6, 10, 20, 50, 100, 200, 500 and 1000 ng/mL. Quality-control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations (8, 420 and 840 ng/mL). The calibration standards and QC samples were stored at −20 ◦ C.
Fig. 2. Mass spectrum of solasodine (a) and midazolam (IS, b).
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J. Ma et al. / J. Chromatogr. B 963 (2014) 24–28
by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) [28,29]. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC samples. The selectivity of the method was evaluated by analyzing six blank rat plasma, blank plasma spiked solasodine and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of solasodine 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 3–1000 ng/mL. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curves, which can be quantified reliably, with an acceptable accuracy (80–120%) and precision (
