Journal of Chromatography B, 990 (2015) 118–124
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of N-methylcytisine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study Shuanghu Wang a,1 , Haiya Wu b,1 , Xueli Huang c , Peiwu Geng a , Congcong Wen c , Jianshe Ma c , Yunfang Zhou a,∗ , Xianqin Wang c,∗∗ a
The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, Wenzhou Medical University, Lishui 323000, China Department of Anesthesiology, Critical Care and Pain Medicine, the Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China c Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 25 December 2014 Received in revised form 21 March 2015 Accepted 27 March 2015 Available online 2 April 2015 Keywords: N-methylcytisine UPLC-MS/MS pharmacokinetics rat plasma
a b s t r a c t In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both < 13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Caulophyllum robustum Maxim., a plant in the Berberidaceae family, is rather ubiquitous throughout northeast, northwest, and southwest China. Its roots have been used in traditional Chinese medicine to treat external injuries and irregular menses [1]. The total alkaloids of Caulophyllum robustum have been demonstrated through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) testing to significantly inhibit proliferation of ECV304 cells, and have been investigated in previous research as a potential anti-tumor, angiogenetic inhibiting drug [2]. Study of the phytochemistry of this plant has revealed the constituent diversity of its pharmacological active alkaloids, such as magnoflorine, taspine, N-methylcytisine (Fig. 1), and
∗ Corresponding author. The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, Wenzhou Medical University, Lishui, Zhejiang 323000, PR China; Tel.: +86 578 2780158. ∗∗ Corresponding author. Analytical and Testing Center, Wenzhou Medical University, Wenzhou, Zhejiang 325035, PR China; Tel.: +86 577 86699156. E-mail addresses: [email protected] (Y. Zhou), [email protected] (X. Wang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2015.03.025 1570-0232/© 2015 Elsevier B.V. All rights reserved.
lupanine [3–6]. Before further pharmacological and pharmacokinetic research is fully possible, it is necessary to first develop analytical methods for the effective determination of Nmethylcytisine in biological fluids. To date, all analytical methods described in previous literature for the determination of N-methylcytisine in biological and other matrices involve either high performance liquid chromatography with ultraviolet detection (HPLC–UV)[7], Evaporative Light Scattering (HPLC–UV)[8], gas chromatography coupled with mass spectrometry (GC-MS)[9], or liquid chromatography-tandem mass spectrometry (LC–MS/MS)[10]. However, N-methylcytisine have low concentration in biological matrices, the limit of detection (LOD) of analytes using these HPLC or GC-MS is not high enough for pharmacokinetic study, and the UPLC-MS/MS which is known to enable increased throughput, sensitivity and better chromatographic peak resolution[11]. No current UPLC–MS/MS method exists for the determination of N-methylcytisine to characterize pharmacokinetic properties. In this study, an ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for determination of N-methylcytisine in rat plasma is developed and successfully applied to pharmacokinetic study of N-methylcytisine after oral and intravenous administration.
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Fig. 1. Mass spectrum of N-methylcytisine (a) and hordenine (IS, b).
2. Experimental 2.1. Chemicals and reagents N-methylcytisine (purity >98%) was a gift from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Hordenine (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). Rat blank plasma samples were supplied from drug-free rats (Laboratory Animal Center of Wenzhou Medical University). 2.2. Instrumentation and conditions A UPLC-MS/MS system with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization (ESI) interface, was used to analyze the compounds. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control. N-methylcytisine and hordenine (IS) were separated using a UPLC BEH HILIC column (2.1 mm × 100 mm, 1.7 m, Waters,
USA) maintained at 40 ◦ C. The initial mobile phase consisted of acetonitrile (containing 10 mM ammonium formate,) and water (containing 0.1% formic acid and 10 mM ammonium formate,) with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 L. Elution was in a linear gradient, where the acetonitrile (containing 10 mM ammonium formate,) content dropped from 90% to 60% between 0 and 1.0 min. The acetonitrile (containing 10 mM ammonium formate,) content was maintained at 60% for 1.0 min, then increased to 90% within 0.5 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including both a strong wash (methanol–water, 50/50, V/V) and a weak wash (methanol–water, 10/90, V/V). Mass spectrometric detection was performed on a triplequadrupole mass spectrometer equipped with an ESI interface in positive mode. Nitrogen was used as the desolvation gas (1000 L/h) and cone gas (50 L/h). Ion monitoring conditions were defined as capillary voltage of 2.5 kV, source temperature of 150 ◦ C, and desolvation temperature of 500 ◦ C. Multiple reaction monitoring (MRM) modes of m/z 205.1→58.0 for N-methylcytisine and m/z 166.1→121.0 for IS were utilized to conduct quantitative analysis. 2.3. Calibration standards and quality control samples The stock solutions of N-methylcytisine (1.0 mg/mL) and hordenine (IS) (100 g/mL) were prepared in methanol-water (50: 50).
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The 0.5 g/mL working standard solution of the IS was prepared from the IS stock solution by dilution with methanol; working solutions for calibration and controls were prepared from stock solutions similarly, using methanol diluent. All of the solutions were stored at 4◦ C and were brought to room temperature before use. N-methylcytisine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were offset to range between 2–2000 ng/mL for N-methylcytisine in rat plasma at 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL, each by adding 10 L of the appropriate working solution to 100 L of blank rat plasma, followed by short vortex mixing. Quality-control (QC) samples were prepared in the same manner as the calibration standards, in three different plasma concentrations (4, 800, and 1600 ng/mL). The calibration standards and QC samples protein precipitation by acetonitrile-methanol (9:1, v/v) before UPLC-MS/MS analysis. 2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. An aliquot of 10 L of the IS working solution (0.5 g/mL) was added to 100 L of the collected plasma sample in a 1.5 mL centrifuge tube, followed by the addition of 200 L of acetonitrilemethanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14900 g for 10 min, the supernatant (2 L) was injected into the UPLC-MS/MS system for analysis. 2.5. Method validation Rigorous tests for selectivity, linearity, accuracy, precision, recovery, and stability, according to the guidelines set by the United States Food and Drug Administration (FDA)[12] and European Medicines Agency (EMA)[13], were conducted in order to thoroughly validate the proposed bioanalytical method. 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 N-methylcytisine and IS, and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of N-methylcytisine-to-IS were plotted against analyte concentrations. Resultant 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 2–2000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, blank rat plasma was extracted and spiked with the analyte at 4, 800, and 1600 ng/mL concentrations (n = 6). The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, this peak area ratio is defined as the matrix effect. The matrix effect of the IS was evaluated at a concentration of 50 ng/mL in a similar manner. Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800, and 1600 ng/mL) over three days of validation testing. The precision is expressed as RSD. The recovery of N-methylcytisine was evaluated by comparing the peak area 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 way. Absolute recovery was evaluated by comparing the peak area of extracted QC samples with compared to neat unextracted standards.
Six aliquots of N-methylcytisine (3000 ng/mL) plasma samples were diluted 10-fold with blank rat plasma for analysis. The peak area of the 3000 ng/ml standard after 10-fold dilution was compared to that of a 300 ng/ml undiluted sample. The acceptance criteria for N-methylcytisine was accuracy (85-115%), and precision (RSD) ≤ 15% according to the six determinations [14]. Carry-over was assessed following injection of a blank plasma sample immediately after 3 repeats of the upper limit of quantification (ULOQ), after which the response was checked for accuracy [15]. Stability values of N-methylcytisine in rat plasma were evaluated by analyzing three replicates of plasma samples at concentrations of 4 or 1600 ng/mL which were all exposed to different conditions. These results were compared with the freshly-prepared plasma samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h, and the ready-to-inject samples (after protein precipitation,) in the HPLC autosampler at room temperature for 24 h. Freeze/thaw stability was evaluated after three complete freeze/thaw cycles (-20 to 25◦ C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at -20◦ C for 20 days. The stability of the IS (50 ng/mL) was evaluated similarly [16,17]. 2.6. Pharmacokinetic study Male Sprague-Dawley rats (200–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University to study the pharmacokinetics of N-methylcytisine. All twelve rats were housed at the Laboratory Animal Center of Wenzhou Medical University. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University, and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine. N-methylcytisine (40 mg) was dissolved in 4 mL saline with little 0.1% HCl, about 3 mL for oral admnistration and 1 mL for intravenous admnistration. The samples were immediately centrifuged at 3000 g for 10 min. The plasma as-obtained (100 L) was stored at −20◦ C until analysis. Plasma N-methylcytisine concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University). The maximum plasma concentration (Cmax ) was observed directly from the concentrationtime curve. The area under the plasma concentration–time curve (AUC) was estimated by the trapezoidal rule. The plasma clearance (CL), apparent volume of distribution (V), and the half-life (t1/2 ) were estimated using non-compartmental calculations performed with DAS software. The absolute bioavailability (Fabs ) is the dosecorrected area under curve (AUC) non-intravenous divided by AUC intravenous. The formula for calculating F for a drug administered by the oral route (po) is given below. Fabs = 100 ×
AUCpo × Doseiv AUCiv × Dosepo
3. Results 3.1. Selectivity and matrix effect Fig. 2 shows typical chromatograms of a blank plasma sample, a blank plasma sample spiked with N-methylcytisine and IS, and a plasma sample. There were no interfering endogenous substances observed at the retention time of the analyte and IS.
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Fig. 2. Representative UPLC-MS/MS chromatograms of N-methylcytisine and hordenine (IS), (a) blank plasma; (b) blank plasma spiked with N-methylcytisine (20 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 8 h after intravenous administration of single dosage 5 mg/kg N-methylcytisine.
The matrix effect for N-methylcytisine at concentrations of 4, 800, and 1600 ng/mL were measured between 95.9% and 98.7% (n = 6). The matrix effect for IS (50 ng/mL) was 102.6% (n = 6). As a result, matrix effect from plasma is considered negligible in this method.
3.2. Calibration curve and sensitivity Linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2–2000 ng/mL for N-methylcytisine in rat plasma. The equation utilized to express the
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calibration curve is: y = 0.00209032*x-0.0246344, r =0.9968, where y represents the ratios of N-methylcytisine peak area to that of IS, and x represents the plasma concentration. The LLOQ for the determination of N-methylcytisine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 11.2% and 92.8%, respectively. The LOD, defined as a signal/noise ratio of 3, was 0.8 ng/mL for N-methylcytisine in rat plasma. 3.3. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over three days of validation tests. Intra-day precision was 10% or less, and inter-day precision was 13% or less at each QC level. The accuracy of the method ranged from 94.5% to 109.4% at each QC level. Mean recoveries of Nmethylcytisine were higher than 86.1%. Absolute recoveries were between 83.2% and 88.6%. The recovery of the IS (50 ng/mL) was 85.6%. Assay performance data is presented below in Table 1. 3.4. Dilution integrity and Carry-over After 10-fold dilution of the six aliquots of N-methylcytisine (3000 ng/mL) samples, the accuracy (n = 6) for N-methylcytisine was identified at 97.8%, and the precision (RSD, n = 6) for Nmethylcytisine was 5.2%. These results met the acceptance criteria for accuracy (85-115%) and precision (RSD) ≤ 15%, suggesting that samples which exceed the calibration curve concentrations can be diluted 10-fold to bring them into the range of the assay with acceptable accuracy and precision. 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 0.5 extra minutes to the end of the gradient elution effectively washed the system between samples, thereby eliminating carry-over [15]. 3.5. Stability Results from the auto-sampler showed that the analyte was stable under room temperature, freeze–thaw, and long-term (20 days) conditions, confirmed because the bias in concentrations were within ±10% of their nominal values (Table 2). To this effect, the established method is suitable for pharmacokinetic study. 3.6. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine is shown in Fig. 3. Primary pharmacokinetic parameters, based on non-compartment model analysis, are summarized in Table 3. The absolute bioavailability of N-methylcytisine was reported as high as 55.5%. 4. Discussion The feasibility of electrospray in positive and negative ion modes of detection was evaluated during the early stages of assay development. Electrospray ionization with positive ion detection resulted in better sensitivity. MS detector parameters were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS-MS conditions, the daughter ion spectrum of the [M + H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most prevalent fragment was detected at m/z 58.0 with a capillary voltage of 2,000 v, and collision energy of 24 v. Accordingly, the m/z 205.1→58.0 transition was selected for further UPLC-MS/MS analysis in MRM mode (Fig. 1).
Fig. 3. Mean plasma concentration time profile after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine in rats.
Liquid chromatographic conditions were developed to separate as many interfering compounds as possible from the analyte and IS. Different columns, such as UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) and UPLC BEH C18 (2.1 mm × 100 mm, 1.7 m) were compared for chromatographic separation. The UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) column demonstrated proper retention time and favorable peak shape for N-methylcytisine and IS over the C18 column. The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization [18–20]. Acetonitrile was selected for the organic phase, as it provides sharper peak shape and lower pump pressure compared to methanol. Acetonitrile (containing 10 mM ammonium formate,) and water (containing 0.1% formic acid and 10 mM ammonium formate,) were chosen as the mobile phase because the combination provides proper retention time and peak shape. The total run time for each injection was 3 min. Efficient removal of proteins and other potential interference in the bio-samples prior to LC-MS analysis was a crucial step in the development of this method [21–23]. An effective and simple protein precipitation was employed in this study. Acetonitrilemethanol (9:1, v/v) was chosen as the protein precipitation solvent because it exhibits acceptable recovery (86.1%-94.8%). Hordenine was selected as the IS, because its chromatographic performance is similar to N-methylcytisine and the two show similar retention time; both are polar substances, as well, and both are suitable for detection in the positive ion electrospray ionization interface. The pharmacokinetic profile of N-methylcytisine in rats was characterized for the first time in this study, and the absolute bioavailability of N-methylcytisine was reported as high as 55.5%, it helps to build a better understanding of the pharmacological features of N-methylcytisine. No current UPLC–MS/MS method exists for the determination of N-methylcytisine to characterize pharmacokinetic properties. Previously, a LC-MS/MS method has sought to determine N-methylcytisine in human urine and herbal samples [10]. A liquid chromatography–tandem mass spectrometry method for simultaneous detection of 22 toxic plant alkaloids, including N-methylcytisine, in herbal and urine samples was developed and validated with run-time of 24 min and LOD of 5 ng/mL. The UPLC-MS/MS method in this study was validated for selectivity, linearity, accuracy, precision, recovery, and stability with a total run time of 3 min–much quicker than previously proposed methods [10].
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Table 1 Precision, accuracy, and recovery for N-methylcytisine of QC samples in rat plasma (n = 6). Concentration (ng/mL)
4 800 1600
RSD (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
9.0 4.4 6.8
12.9 8.7 8.9
101.2 94.5 105.8
109.4 104.9 95.6
Recovery (%)
Absolute recovery (%)
93.1 86.1 94.8
85.6 83.2 88.6
Table 2 Summary of stability of N-methylcytisine and IS under various storage conditions (n = 3). Condition
Concentration (ng/mL) Added
Measured
Ambient, 2 h
4 1600 (IS)50 4 1600 (IS)50 4 1600 (IS)50 4 1600 (IS)50
4.2 1607.3 49.6 3.7 1750.6 52.7 4.3 1503.2 48.6 3.9 1578.2 51.2
−20 ◦ C, 20 days
3 freeze thaw
Autosampler ambient 24 h
RSD (%)
Accuracy (%)
3.4 1.7 0.6 11.6 7.6 6.2 7.9 8.7 9.6 1.7 1.3 2.5
105.0 100.5 99.2 92.5 109.4 105.4 107.5 94.0 97.2 98.8 98.6 102.4
Table 3 Primary pharmacokinetic parameters after oral or intravenous administration of N-methylcytisine in rats (n = 6). Parameters
Unit
AUC(0–t) AUC(0–∞) t1/2 CL V Cmax Absolute bioavailability/Fabs
ng/mL h ng/mL h h L/h/kg L/kg ng/mL
Mean
SD
Mean
568.5 597.0 0.5 0.03 0.5 425.7
13082.0 13459.8 4.6 0.37 2.5 1779.4
po 10 mg/kg 14514.0 14744.2 4.1 0.68 4.0 2619.0 55.5%
5. Conclusion The UPLC-MS/MS method described here allows accurate and easily reproduced determination of N-methylcytisine in rat plasma, utilizing 100 L of plasma with an LLOQ of 2 ng/mL. The UPLCMS/MS method was successfully applied to a pharmacokinetic study of N-methylcytisine after both oral and intravenous administration. Notably, the pharmacokinetic profile of N-methylcytisine in rats was characterized for the first time. The absolute bioavailability of N-methylcytisine was identified at 55.5% for first time, as well.
Acknowledgements This work was supported by fund of the National Natural Science Foundation of China, No. 81401558.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of N-methylcytisine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study Shuanghu Wang a,1 , Haiya Wu b,1 , Xueli Huang c , Peiwu Geng a , Congcong Wen c , Jianshe Ma c , Yunfang Zhou a,∗ , Xianqin Wang c,∗∗ a
The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, Wenzhou Medical University, Lishui 323000, China Department of Anesthesiology, Critical Care and Pain Medicine, the Second Affiliated Hospital, Wenzhou Medical University, Wenzhou 325000, China c Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325035, China b
a r t i c l e
i n f o
Article history: Received 25 December 2014 Received in revised form 21 March 2015 Accepted 27 March 2015 Available online 2 April 2015 Keywords: N-methylcytisine UPLC-MS/MS pharmacokinetics rat plasma
a b s t r a c t In this work, a sensitive and selective UPLC-MS/MS method for determination of N-methylcytisine in rat plasma is developed. After addition of hordenine as an internal standard (IS), protein precipitation by acetonitrile-methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) with acetonitrile (containing 10 mM ammonium formate) and water (containing 0.1% formic acid and 10 mM ammonium formate) as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 205.1→58.0 for N-methylcytisine, and m/z 166.1→121.0 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for N-methylcytisine in rat plasma. Mean recoveries of N-methylcytisine in rat plasma ranged from 86.1% to 94.8%. RSD of intra-day and inter-day precision were both < 13%. The accuracy of the method was between 94.5% and 109.4%. The method was successfully applied to pharmacokinetic study of N-methylcytisine after either oral or intravenous administration. For the first time, the absolute bioavailability of N-methylcytisine was reported as high as 55.5%. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Caulophyllum robustum Maxim., a plant in the Berberidaceae family, is rather ubiquitous throughout northeast, northwest, and southwest China. Its roots have been used in traditional Chinese medicine to treat external injuries and irregular menses [1]. The total alkaloids of Caulophyllum robustum have been demonstrated through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) testing to significantly inhibit proliferation of ECV304 cells, and have been investigated in previous research as a potential anti-tumor, angiogenetic inhibiting drug [2]. Study of the phytochemistry of this plant has revealed the constituent diversity of its pharmacological active alkaloids, such as magnoflorine, taspine, N-methylcytisine (Fig. 1), and
∗ Corresponding author. The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui, Wenzhou Medical University, Lishui, Zhejiang 323000, PR China; Tel.: +86 578 2780158. ∗∗ Corresponding author. Analytical and Testing Center, Wenzhou Medical University, Wenzhou, Zhejiang 325035, PR China; Tel.: +86 577 86699156. E-mail addresses: [email protected] (Y. Zhou), [email protected] (X. Wang). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2015.03.025 1570-0232/© 2015 Elsevier B.V. All rights reserved.
lupanine [3–6]. Before further pharmacological and pharmacokinetic research is fully possible, it is necessary to first develop analytical methods for the effective determination of Nmethylcytisine in biological fluids. To date, all analytical methods described in previous literature for the determination of N-methylcytisine in biological and other matrices involve either high performance liquid chromatography with ultraviolet detection (HPLC–UV)[7], Evaporative Light Scattering (HPLC–UV)[8], gas chromatography coupled with mass spectrometry (GC-MS)[9], or liquid chromatography-tandem mass spectrometry (LC–MS/MS)[10]. However, N-methylcytisine have low concentration in biological matrices, the limit of detection (LOD) of analytes using these HPLC or GC-MS is not high enough for pharmacokinetic study, and the UPLC-MS/MS which is known to enable increased throughput, sensitivity and better chromatographic peak resolution[11]. No current UPLC–MS/MS method exists for the determination of N-methylcytisine to characterize pharmacokinetic properties. In this study, an ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method for determination of N-methylcytisine in rat plasma is developed and successfully applied to pharmacokinetic study of N-methylcytisine after oral and intravenous administration.
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Fig. 1. Mass spectrum of N-methylcytisine (a) and hordenine (IS, b).
2. Experimental 2.1. Chemicals and reagents N-methylcytisine (purity >98%) was a gift from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Hordenine (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). Rat blank plasma samples were supplied from drug-free rats (Laboratory Animal Center of Wenzhou Medical University). 2.2. Instrumentation and conditions A UPLC-MS/MS system with ACQUITY I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization (ESI) interface, was used to analyze the compounds. The UPLC system was comprised of a Binary Solvent Manager (BSM) and a Sample Manager with Flow-Through Needle (SM-FTN). Masslynx 4.1 software (Waters Corp.) was used for data acquisition and instrument control. N-methylcytisine and hordenine (IS) were separated using a UPLC BEH HILIC column (2.1 mm × 100 mm, 1.7 m, Waters,
USA) maintained at 40 ◦ C. The initial mobile phase consisted of acetonitrile (containing 10 mM ammonium formate,) and water (containing 0.1% formic acid and 10 mM ammonium formate,) with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 L. Elution was in a linear gradient, where the acetonitrile (containing 10 mM ammonium formate,) content dropped from 90% to 60% between 0 and 1.0 min. The acetonitrile (containing 10 mM ammonium formate,) content was maintained at 60% for 1.0 min, then increased to 90% within 0.5 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including both a strong wash (methanol–water, 50/50, V/V) and a weak wash (methanol–water, 10/90, V/V). Mass spectrometric detection was performed on a triplequadrupole mass spectrometer equipped with an ESI interface in positive mode. Nitrogen was used as the desolvation gas (1000 L/h) and cone gas (50 L/h). Ion monitoring conditions were defined as capillary voltage of 2.5 kV, source temperature of 150 ◦ C, and desolvation temperature of 500 ◦ C. Multiple reaction monitoring (MRM) modes of m/z 205.1→58.0 for N-methylcytisine and m/z 166.1→121.0 for IS were utilized to conduct quantitative analysis. 2.3. Calibration standards and quality control samples The stock solutions of N-methylcytisine (1.0 mg/mL) and hordenine (IS) (100 g/mL) were prepared in methanol-water (50: 50).
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The 0.5 g/mL working standard solution of the IS was prepared from the IS stock solution by dilution with methanol; working solutions for calibration and controls were prepared from stock solutions similarly, using methanol diluent. All of the solutions were stored at 4◦ C and were brought to room temperature before use. N-methylcytisine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were offset to range between 2–2000 ng/mL for N-methylcytisine in rat plasma at 2, 5, 10, 20, 50, 100, 200, 500, 1000, and 2000 ng/mL, each by adding 10 L of the appropriate working solution to 100 L of blank rat plasma, followed by short vortex mixing. Quality-control (QC) samples were prepared in the same manner as the calibration standards, in three different plasma concentrations (4, 800, and 1600 ng/mL). The calibration standards and QC samples protein precipitation by acetonitrile-methanol (9:1, v/v) before UPLC-MS/MS analysis. 2.4. Sample preparation Before analysis, the plasma sample was thawed to room temperature. An aliquot of 10 L of the IS working solution (0.5 g/mL) was added to 100 L of the collected plasma sample in a 1.5 mL centrifuge tube, followed by the addition of 200 L of acetonitrilemethanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14900 g for 10 min, the supernatant (2 L) was injected into the UPLC-MS/MS system for analysis. 2.5. Method validation Rigorous tests for selectivity, linearity, accuracy, precision, recovery, and stability, according to the guidelines set by the United States Food and Drug Administration (FDA)[12] and European Medicines Agency (EMA)[13], were conducted in order to thoroughly validate the proposed bioanalytical method. 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 N-methylcytisine and IS, and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of N-methylcytisine-to-IS were plotted against analyte concentrations. Resultant 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 2–2000 ng/mL. The LLOQ was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, blank rat plasma was extracted and spiked with the analyte at 4, 800, and 1600 ng/mL concentrations (n = 6). The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations, this peak area ratio is defined as the matrix effect. The matrix effect of the IS was evaluated at a concentration of 50 ng/mL in a similar manner. Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800, and 1600 ng/mL) over three days of validation testing. The precision is expressed as RSD. The recovery of N-methylcytisine was evaluated by comparing the peak area 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 way. Absolute recovery was evaluated by comparing the peak area of extracted QC samples with compared to neat unextracted standards.
Six aliquots of N-methylcytisine (3000 ng/mL) plasma samples were diluted 10-fold with blank rat plasma for analysis. The peak area of the 3000 ng/ml standard after 10-fold dilution was compared to that of a 300 ng/ml undiluted sample. The acceptance criteria for N-methylcytisine was accuracy (85-115%), and precision (RSD) ≤ 15% according to the six determinations [14]. Carry-over was assessed following injection of a blank plasma sample immediately after 3 repeats of the upper limit of quantification (ULOQ), after which the response was checked for accuracy [15]. Stability values of N-methylcytisine in rat plasma were evaluated by analyzing three replicates of plasma samples at concentrations of 4 or 1600 ng/mL which were all exposed to different conditions. These results were compared with the freshly-prepared plasma samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h, and the ready-to-inject samples (after protein precipitation,) in the HPLC autosampler at room temperature for 24 h. Freeze/thaw stability was evaluated after three complete freeze/thaw cycles (-20 to 25◦ C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at -20◦ C for 20 days. The stability of the IS (50 ng/mL) was evaluated similarly [16,17]. 2.6. Pharmacokinetic study Male Sprague-Dawley rats (200–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University to study the pharmacokinetics of N-methylcytisine. All twelve rats were housed at the Laboratory Animal Center of Wenzhou Medical University. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University, and were in accordance with the Guide for the Care and Use of Laboratory Animals. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine. N-methylcytisine (40 mg) was dissolved in 4 mL saline with little 0.1% HCl, about 3 mL for oral admnistration and 1 mL for intravenous admnistration. The samples were immediately centrifuged at 3000 g for 10 min. The plasma as-obtained (100 L) was stored at −20◦ C until analysis. Plasma N-methylcytisine concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University). The maximum plasma concentration (Cmax ) was observed directly from the concentrationtime curve. The area under the plasma concentration–time curve (AUC) was estimated by the trapezoidal rule. The plasma clearance (CL), apparent volume of distribution (V), and the half-life (t1/2 ) were estimated using non-compartmental calculations performed with DAS software. The absolute bioavailability (Fabs ) is the dosecorrected area under curve (AUC) non-intravenous divided by AUC intravenous. The formula for calculating F for a drug administered by the oral route (po) is given below. Fabs = 100 ×
AUCpo × Doseiv AUCiv × Dosepo
3. Results 3.1. Selectivity and matrix effect Fig. 2 shows typical chromatograms of a blank plasma sample, a blank plasma sample spiked with N-methylcytisine and IS, and a plasma sample. There were no interfering endogenous substances observed at the retention time of the analyte and IS.
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Fig. 2. Representative UPLC-MS/MS chromatograms of N-methylcytisine and hordenine (IS), (a) blank plasma; (b) blank plasma spiked with N-methylcytisine (20 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 8 h after intravenous administration of single dosage 5 mg/kg N-methylcytisine.
The matrix effect for N-methylcytisine at concentrations of 4, 800, and 1600 ng/mL were measured between 95.9% and 98.7% (n = 6). The matrix effect for IS (50 ng/mL) was 102.6% (n = 6). As a result, matrix effect from plasma is considered negligible in this method.
3.2. Calibration curve and sensitivity Linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 2–2000 ng/mL for N-methylcytisine in rat plasma. The equation utilized to express the
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calibration curve is: y = 0.00209032*x-0.0246344, r =0.9968, where y represents the ratios of N-methylcytisine peak area to that of IS, and x represents the plasma concentration. The LLOQ for the determination of N-methylcytisine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 11.2% and 92.8%, respectively. The LOD, defined as a signal/noise ratio of 3, was 0.8 ng/mL for N-methylcytisine in rat plasma. 3.3. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over three days of validation tests. Intra-day precision was 10% or less, and inter-day precision was 13% or less at each QC level. The accuracy of the method ranged from 94.5% to 109.4% at each QC level. Mean recoveries of Nmethylcytisine were higher than 86.1%. Absolute recoveries were between 83.2% and 88.6%. The recovery of the IS (50 ng/mL) was 85.6%. Assay performance data is presented below in Table 1. 3.4. Dilution integrity and Carry-over After 10-fold dilution of the six aliquots of N-methylcytisine (3000 ng/mL) samples, the accuracy (n = 6) for N-methylcytisine was identified at 97.8%, and the precision (RSD, n = 6) for Nmethylcytisine was 5.2%. These results met the acceptance criteria for accuracy (85-115%) and precision (RSD) ≤ 15%, suggesting that samples which exceed the calibration curve concentrations can be diluted 10-fold to bring them into the range of the assay with acceptable accuracy and precision. 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 0.5 extra minutes to the end of the gradient elution effectively washed the system between samples, thereby eliminating carry-over [15]. 3.5. Stability Results from the auto-sampler showed that the analyte was stable under room temperature, freeze–thaw, and long-term (20 days) conditions, confirmed because the bias in concentrations were within ±10% of their nominal values (Table 2). To this effect, the established method is suitable for pharmacokinetic study. 3.6. Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine is shown in Fig. 3. Primary pharmacokinetic parameters, based on non-compartment model analysis, are summarized in Table 3. The absolute bioavailability of N-methylcytisine was reported as high as 55.5%. 4. Discussion The feasibility of electrospray in positive and negative ion modes of detection was evaluated during the early stages of assay development. Electrospray ionization with positive ion detection resulted in better sensitivity. MS detector parameters were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS-MS conditions, the daughter ion spectrum of the [M + H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most prevalent fragment was detected at m/z 58.0 with a capillary voltage of 2,000 v, and collision energy of 24 v. Accordingly, the m/z 205.1→58.0 transition was selected for further UPLC-MS/MS analysis in MRM mode (Fig. 1).
Fig. 3. Mean plasma concentration time profile after oral (10 mg/kg) or intravenous (5 mg/kg) administration of N-methylcytisine in rats.
Liquid chromatographic conditions were developed to separate as many interfering compounds as possible from the analyte and IS. Different columns, such as UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) and UPLC BEH C18 (2.1 mm × 100 mm, 1.7 m) were compared for chromatographic separation. The UPLC BEH HILIC (2.1 mm × 100 mm, 1.7 m) column demonstrated proper retention time and favorable peak shape for N-methylcytisine and IS over the C18 column. The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization [18–20]. Acetonitrile was selected for the organic phase, as it provides sharper peak shape and lower pump pressure compared to methanol. Acetonitrile (containing 10 mM ammonium formate,) and water (containing 0.1% formic acid and 10 mM ammonium formate,) were chosen as the mobile phase because the combination provides proper retention time and peak shape. The total run time for each injection was 3 min. Efficient removal of proteins and other potential interference in the bio-samples prior to LC-MS analysis was a crucial step in the development of this method [21–23]. An effective and simple protein precipitation was employed in this study. Acetonitrilemethanol (9:1, v/v) was chosen as the protein precipitation solvent because it exhibits acceptable recovery (86.1%-94.8%). Hordenine was selected as the IS, because its chromatographic performance is similar to N-methylcytisine and the two show similar retention time; both are polar substances, as well, and both are suitable for detection in the positive ion electrospray ionization interface. The pharmacokinetic profile of N-methylcytisine in rats was characterized for the first time in this study, and the absolute bioavailability of N-methylcytisine was reported as high as 55.5%, it helps to build a better understanding of the pharmacological features of N-methylcytisine. No current UPLC–MS/MS method exists for the determination of N-methylcytisine to characterize pharmacokinetic properties. Previously, a LC-MS/MS method has sought to determine N-methylcytisine in human urine and herbal samples [10]. A liquid chromatography–tandem mass spectrometry method for simultaneous detection of 22 toxic plant alkaloids, including N-methylcytisine, in herbal and urine samples was developed and validated with run-time of 24 min and LOD of 5 ng/mL. The UPLC-MS/MS method in this study was validated for selectivity, linearity, accuracy, precision, recovery, and stability with a total run time of 3 min–much quicker than previously proposed methods [10].
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Table 1 Precision, accuracy, and recovery for N-methylcytisine of QC samples in rat plasma (n = 6). Concentration (ng/mL)
4 800 1600
RSD (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
9.0 4.4 6.8
12.9 8.7 8.9
101.2 94.5 105.8
109.4 104.9 95.6
Recovery (%)
Absolute recovery (%)
93.1 86.1 94.8
85.6 83.2 88.6
Table 2 Summary of stability of N-methylcytisine and IS under various storage conditions (n = 3). Condition
Concentration (ng/mL) Added
Measured
Ambient, 2 h
4 1600 (IS)50 4 1600 (IS)50 4 1600 (IS)50 4 1600 (IS)50
4.2 1607.3 49.6 3.7 1750.6 52.7 4.3 1503.2 48.6 3.9 1578.2 51.2
−20 ◦ C, 20 days
3 freeze thaw
Autosampler ambient 24 h
RSD (%)
Accuracy (%)
3.4 1.7 0.6 11.6 7.6 6.2 7.9 8.7 9.6 1.7 1.3 2.5
105.0 100.5 99.2 92.5 109.4 105.4 107.5 94.0 97.2 98.8 98.6 102.4
Table 3 Primary pharmacokinetic parameters after oral or intravenous administration of N-methylcytisine in rats (n = 6). Parameters
Unit
AUC(0–t) AUC(0–∞) t1/2 CL V Cmax Absolute bioavailability/Fabs
ng/mL h ng/mL h h L/h/kg L/kg ng/mL
Mean
SD
Mean
568.5 597.0 0.5 0.03 0.5 425.7
13082.0 13459.8 4.6 0.37 2.5 1779.4
po 10 mg/kg 14514.0 14744.2 4.1 0.68 4.0 2619.0 55.5%
5. Conclusion The UPLC-MS/MS method described here allows accurate and easily reproduced determination of N-methylcytisine in rat plasma, utilizing 100 L of plasma with an LLOQ of 2 ng/mL. The UPLCMS/MS method was successfully applied to a pharmacokinetic study of N-methylcytisine after both oral and intravenous administration. Notably, the pharmacokinetic profile of N-methylcytisine in rats was characterized for the first time. The absolute bioavailability of N-methylcytisine was identified at 55.5% for first time, as well.
Acknowledgements This work was supported by fund of the National Natural Science Foundation of China, No. 81401558.
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