Drug Testing and Analysis
Short communication Received: 3 March 2015
Revised: 17 April 2015
Accepted: 1 May 2015
Published online in Wiley Online Library: 20 May 2015
(www.drugtestinganalysis.com) DOI 10.1002/dta.1819
Simultaneous quantification of phencynonate and its active metabolite N-demethyl phencynonate in human plasma using liquid chromatography and isotope-dilution mass spectrometry Zhengang Chen, Hui Xie, Jinbo Liu and Guangshun Wang* A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously quantify phencynonate (PCN) and its major metabolite N-demethyl phencynonate (DM-PCN) in human plasma. Following one-step liquid-liquid extraction, the analytes were separated on a reversed-phase C18 column. Methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was used as isocratic mobile phase at a flow-rate of 0.3 mL/min. An API 5000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. Multiple reaction monitoring using the transition of m/z 358.4 → m/z 156.2, m/z 344.4 → m/z 142.2, and m/z 361.3 → m/z 159.2 was performed to quantify PCN, DM-PCN, and the internal standard (D3-PCN), respectively. This approach showed a lower limit of quantification of 10 pg/mL and 25 pg/mL for PCN and DM-PCN in plasma, respectively. This sensitivity was at least 50-fold superior to previously reported ones and thus enabled the approach well applicable to low-dose pharmacokinetic studies. The intra- and inter-day precisions were less than 14.2 % at each QC level for both PCN and DM-PCN. The inter-day relative errors ranged from -1.9% to -4.9% for PCN, and from 0.6% to 6.4% for DM-PCN. As a proof of principle, the validated method was successfully applied to simultaneous quantification of circulating PCN and DM-PCN in healthy subjects after a single oral administration of 2 mg phencynonate hydrochloride pellet. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: phencynonate; N-demethyl phencynonote; isotope-dilution LC-MS; clinic pharmacokinetics
Introduction
Experimental
Phencynonate (PCN, 3-Methyl-3-azabicyclo(3,3,1)nonanyl-9-α-yl-αcyclopentyl-α-phenyl-α-glycolate, Figure 1) is an anti-cholinergic agent showing therapeutic effects towards motion sickness, epilepsy and Parkinson’s disease.[1] PCN undergoes hepatic dealkylation and converts to its active metabolite N-demethyl phencynonate in vivo (DM-PCN, Figure 1).[2–4] The pre-clinical pharmacokinetics, distribution, and metabolic pathways of PCN in experimental animals have been well described. Additionally, several approaches were developed to quantify PCN or its analogues in the body fluids/tissues of experimental animals.[2,5–8] Kou et al. reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to determine PCN enantiomers in rat blood with a low limit of quantification (LLOQ) of 1.0 ng/mL.[5] Li et al. reported an LC-MS/MS method to quantify PCN in rat plasma with an LLOQ of 0.5 ng/mL.[7] However, the human pharmacokinetics of PCN and its major metabolite DM-PCN have not been demonstrated to date. As the clinical dose of PCN is as low as 2 mg, a sensitive method is thus essential for the human pharmacokinetics study. In this work, a sensitive and selective LC-MS/MS method was developed and validated to quantify PCN and DM-PCN simultaneously in human plasma. This method was at least 50-fold more sensitive than previously reported ones and was well applicable to low-dose clinical pharmacokinetic studies.
Chemicals and reagents
LC–MS/MS The liquid chromatography system (Agilent, Santa Clara, CA, USA) was equipped with an LC-G1312A pump system and a G1367B autosampler. The chromatographic separation was achieved on an Agela C18 column (50 × 2.1 mm, 5 μm, Agela, Tianjin, China) at 25°C. The mobile phase was consisted of methanol and 0.02%
* Correspondence to: Guangshun Wang, Bao Di Clinical College of Tianjin Medical University, No.8 Guang Chuan Road, Baodi District, Tianjin, China. E-mail: [email protected] Bao Di Clinical College of Tianjin Medical University, Tianjin 301800, China
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Drug Test. Analysis 2015, 7, 843–847
Phencynonate hydrochloride was provided by Beijin Hwells Pharmaceuticals (Beijing, China). D3-phencynonate (internal standard, IS) was purchased from High Standard Products Corporation (Westminster, CA, USA). DM-PCN was provided by Institute of Pharmacology & Toxicology of AMMS (Beijing, China). HPLC-grade acetonitrile and methanol were purchased from Dikma Technologies Inc. (Lake Forest, CA, USA). All other reagents were of analytical grade.
Drug Testing and Analysis
Z. Chen et al. Method validation
Figure 1. Chemical structures of (A) PCN, (B) DM-PCN and (C) D3-PCN (internal standard).
formic acid in 10 mM ammonium acetate (62:38, v/v) and run at a flow-rate of 0.3 mL/min. Each run time was 4 min. Mass spectrometric detection was performed on an API 5000 triple quadrupole instrument equipped with a TurboIonspray interface (AB SCIEX, Toronto, Canada). The data was acquired and processed using Analyst 1.5 software. Multiple reaction monitoring (MRM) analysis was applied in a positive ionization mode to detect precursor to product ion transitions at m/z 358.4 → m/z 156.2 with CE equal to 22 eV for PCN, m/z 344.4 →m/z 142.2 with CE equal to 30 eV for DM-PCN , and m/z 361.3 → m/z 159.2 with CE equal to 22 eV for IS. The ion spray voltage set at +5.5 kV and the heater gas temperature set to 600°C. The nebulizer gas (Gas 1) set at 0.36 MPa, a heater gas (Gas 2) of 0.31 MPa, a curtain gas of 0.069 MPa, and a collision gas of 0.055 MPa.
Sample preparation Once thawed, the plasma samples were vortexed and 200 μL plasma samples were taken. The samples were spiked with 25 μL of the working IS solution, 20 μL of an acetonitrile–water solution (50:50, v/v), 100 μL of methanol, and 20 μL of 20% ZnSO4 aqueous solution. The mixture was then vortex-mixed for 30 s and added to 1 mL of ethyl acetate. Finally, the mixture was vortexed for 30 s, twice. The two phases were separated through centrifugation at 3000 × g for 10 min at room temperature. The upper organic layer was transferred into another tube and completely evaporated under a stream of nitrogen. The residue was reconstituted with 200 μL of acetonitrile–water (50:50, v/v) and a 10-μL aliquot of the reconstituted extract was injected into LC–MS/MS system.
Standard, QC, and IS preparation
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Primary stock solutions of PCN at 0.994 mg/mL and DM-PCN at 0.519 mg/mL were prepared by dissolving accurately weighed amounts of the reference substances in acetonitrile–water (50:50, v/v). The stock solutions were serially diluted with acetonitrile– water (50:50, v/v) to give working solutions at the following concentrations for PCN and DM-PCN: 0.1/0.25, 0.2/0.5, 0.5/1.25, 1.25/3.13, 5/12.5, 12.5/31.3, 25/62.5, 40/100, and 50/125 ng/mL. Two other stock solutions were independently prepared and diluted with human plasma to achieve QC samples at concentrations of 30/75 pg/mL (LQC), 1500/3750 pg/mL (MQC), and 3500/8750 pg/mL (HQC) for PCN and DM-PCN. IS solution (100 ng/mL) was prepared by diluting the 8 mg/mL D3PCN stock solution with the acetonitrile–water mixture (50:50, v/v). All solutions were kept at 4°C and brought to room temperature before use. QC samples were kept at –70°C, brought to room temperature until completely thawed, and then vortexed before use. The calibration standards were prepared by spiking 200 μL blank plasma with 20 μL of the corresponding working solutions.
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The method was validated by verifying specificity, linearity, precision and accuracy, recovery, matrix effect, and stability. The method validation was performed according to FDA guidelines for Bioanalytical Method Validation.[9] Method specificity was evaluated by assaying human blank plasma samples from six different donors and LLOQ samples. LLOQ is defined as the lowest concentration of the analyte that is at least 5 times the response compared to blank response and that can be determined with acceptable precision and accuracy [six replicates in three validation days with a relative standard deviation (RSD) below 20% and a relative error (RE) within ±20%]. Linearity was assessed by plotting calibration curves in human plasma duplicated in three separate runs. The curves were fitted by a linear weighted (1/x2) least squares regression method through measurement of the peak-area ratios of the analytes to the IS solution. To evaluate the precision and accuracy of the method, QC samples at three concentration levels (30, 1500, and 3500 pg/mL for PCN, and 75, 3750, and 8750 pg/mL for DM-PCN) were analyzed in six replicates in five different batches within three validation days. The RSD determined assay precision, along with one-way analysis of variance. As well, the RSD sorted out sources of variance due to within- and between-run factors. Assay accuracy was expressed as the RE, or (observed concentration – nominal concentration) / (nominal concentration) × 100 %. The required accuracy was within ± 15 %, and the intra- and inter-day precision values were not to exceed 15 %. The recovery of the extraction procedure was estimated at three concentration levels (30, 1500, and 3500 pg/mL for PCN, and 75, 3750, and 8750 pg/mL for DM-PCN) by comparing the peak areas of blank samples spiked before and after liquid-liquid extraction (LLE). Spike-after-extraction samples represented 100% recovery. Matrix effects were assessed to determine whether potential ion suppression or enhancement due to the co-elution of matrix components existed in the experiment. The corresponding peak areas of the analytes from the spike-after-extraction samples at low, medium, and high concentration levels were compared to the standard solution at the same concentration in the mobile phase. The variability in the values, expressed as RSD (%), was a measure of the relative matrix effect for the analyte, which should be less than 15%. The stability of PCN and DM-PCN in human plasma was evaluated by analyzing replicates (n = 3) of plasma samples exposed to different conditions (time and temperature) at 30, 1500, 3500 pg/mL for PCN, and 75, 375, and 875 pg/mL for DM-PCN. These results were compared to those obtained from freshly prepared plasma samples. The analytes were considered to be stable in the biological matrix when 85–115 % of the initial concentration was retained.
Clinical application The developed method was applied to determine the plasma concentrations of PCN and DM-PCN. The study was approved by the Ethics Committee of Baodi Hospital. A total of 10 healthy male Chinese volunteers were included in the study. Blood samples were drawn, through a sodium citrate anticoagulative tube, at the baseline (before drug administration) and at 0.25, 0.50, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 12, 24, 36, and 48 h after oral administration of 2 mg PCN tablet. Plasma samples were obtained by centrifugation at 2000 × g for 10 min and frozen at –70°C until analysis.
Copyright © 2015 John Wiley & Sons, Ltd.
Drug Test. Analysis 2015, 7, 843–847
Drug Testing and Analysis
LC/MS quantifiaciton of phencynonate and N-demethyl phencynonate Time profiles of plasma concentrations of PCN and DM-PCN were acquired for each subject. The major non-compartmental pharmacokinetic parameters of PCN and DM-PCN were then calculated. The maximum plasma concentration (Cmax) and the time of occurrence (Tmax) were obtained directly from the measured data. The terminal elimination rate constant (ke) was estimated by linear least-squares regression of the terminal portion of the plasma concentration–time curve, and the corresponding elimination half-life (T1/2) was then calculated as 0.693/ke. The area under the plasma concentration-time curve (AUC) was calculated according to the linear trapezoidal rule to the last measurable point (AUC0 t) or to infinity (AUC0 ∞) by AUC0 t + Ct/ke, where Ct was the last measurable drug concentration.
bond. Accordingly, an MRM transition channel of m/z 358.4 → m/z 156.2 was chosen to quantify PCN. A transition of m/z 344.4 → m/z 142.2 was chosen for DM-PCN. Deuterated PCN (D3-PCN) was chosen as internal standard to further improve the assay accuracy and precision. The corresponding transition of IS was set as m/z 361.3 → m/z 159.2. A variety of mobile phase solvents and additives were investigated and evaluated in terms of both sensitivity and separation efficiency. An isocratic elution using methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was finally employed. The optimal flow rate was set at 0.3 mL/min. No appreciable interferences from plasma were observed at the retention times of targeted analytes (Figure 2). Method validation Linearity of calibration standards
Results and discussion Optimization of mass spectrometric and chromatographic conditions Low dose PCN (e.g. 2 mg/day) substantially attenuates the adverse effects of anti-cholinergics in clinic applications, albeit poses a daunting challenge for pharmacokinetic study. To achieve the necessary sensitivity (pg/mL level), the LC-MS conditions were systemically optimized as described below. Electrospray ionization (ESI) at positive ionization mode was selected for the quantification of PCN and DM-PCN. Under the experiment conditions, soft ionization in the ESI source produced the protonated molecules [M + H]+ of PCN and DM-PCN at m/z 358 and m/z 344 as the base peak, respectively. The product ion mass spectrum of [M + H]+ of PCN showed a major fragment at m/z 156 resulting from the cleavage of the ester
Plotted calibration curves and correlation coefficients > 0.99 confirm that the calibration curves were linear over the concentration range 10 5000 pg/mL and 25 12500 pg/mL for PCN and DM-PCN, respectively. Typical curves for PCN and DM-PCN are as follows: y = 3.44 × 10 3 x 1.71 × 10 3, r = 0.9963, and y = 1.10 × 10 3 x 6.26×10 4, r = 0.9979, where y represents the ratio of the analyte peak area to that of IS and x is the plasma concentration of the analyte. Assay specificity and LLOQ Three typical MRM chromatograms from the study of PCN and DM-PCN in human plasma are shown in Figure 2. No interfering peaks were observed in blank plasma (Figure 2A). A representative MRM chromatogram of blank plasma spiked with PCN (10 pg/mL),
Drug Test. Analysis 2015, 7, 843–847
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 2. Representative chromatograms for PCN (I), DM-PCN (II), and D3-PCN (IS, III) in human plasma samples. (A) the MRM chromatograms of the blank plasma sample; (B) the MRM chromatograms of blank plasma spiked with PCN (10 pg/mL), DM-PCN (25 pg/mL), and D3-PCN (500 pg/mL); (C) the MRM chromatograms of a plasma sample taken from a volunteer 5 h after oral administration of 2 mg PCN.
Drug Testing and Analysis
Z. Chen et al.
DM-PCN (25 pg/mL) and IS (500 pg/mL) is shown in Figure 2B. A sample from a volunteer 0.5 h after oral administration of 2 mg PCN is shown in Figure 2C. For PCN, DM-PCN, and IS, the chromatograms were free from endogenous matrix interference at their respective retention times. For PCN and DM-PCN, the LC-MS/MS method yielded an LLOQ of 10 pg/mL and 25 pg/mL with accuracies of –1.32% and 2.5% in terms of RE; the precision values were less than 3.9% and 10.4% in terms of RSD, respectively. The data are shown in Table 1. The signal-to-noise ratio (S/N) of the LLOQ was 22.5 for PCN and 12.8 for DM-PCN, respectively (as shown in Figure 2B). Precision and accuracy The intra- and inter-day precision and accuracy were calculated by analysis of variance based on replicate analyses (n = 6) of QC samples. In this study, the intra- and inter-day precision values were less than 12.6% and 14.2% for each QC level of PCN and DM-PCN, respectively. The inter-day relative errors were –1.9%, –2.9%, and –4.9%, and 6.0%, 6.4%, and 0.6% for low, medium, and high concentrations of PCN and DM-PCN, respectively. These data indicate reproducible LC-MS/MS results and prove that the assay was accurate and reliable. The accuracy and precision data are shown in Table 1. Recovery The extraction recovery of PCN determined at concentrations of 30, 1500, and 3500 pg/mL were 93.3%, 105%, and 107%, respectively. The extraction recovery of DM-PCN determined at concentrations of 75, 3750, and 8750 pg/mL were 72.5%, 79.5%, 84.1%, respectively. The extraction recovery of IS was determined to be 99.4%. Matrix effect The matrix factors of PCN determined at concentrations of 30, 1500, and 3500 pg/mL were 115%, 104%, and 102%, respectively, and the RSD values from six lots of plasma were less than 4.7%. The matrix factors of PCN determined at concentrations of 75, 3750, and 8750 pg/mL were 111%, 99.9%, and 93.6%, respectively, and the RSD values from six lots of plasma were less than 4.5%. Stability For both PCN and DM-PCN, the plasma samples were stable for 6 h at 25°C, 7 h after preparation at 25°C, 53 h after preparation at 4°C and after 4 freeze-thaw cycles ( 70 to 25°C) on consecutive days; the RE values were within ±15 % for all low, media, and high Table 1. Accuracy and precision for the analysis of PCN and DM-PCN in human plasma Compound
PCN
DM-PCN
Nominal Mean Relative Intra-day Inter-day plasma measured error RSD RSD concentration concentration
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(pg/mL)
(pg/mL)
10 30 1500 3500 25 75 3750 8750
9.87 29.4 1457 3329 24.7 79.5 3992 8805
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(%) 1.3 1.9 2.9 4.9 2.5 6.0 6.4 0.6
(%)
(%)
3.9 6.6 2.7 4.0 10.4 7.0 5.9 8.9
12.6 11.0 12.2 13.6 12.1 14.2
Figure 3. Mean plasma concentration-time curve of PCN and DM-PCN in healthy subjects (n = 10, mean ± SD).
Table 2. The main pharmacokinetic parameters of PCN and DM-PCN after an oral administration of 2 mg PCN hydrochloride to ten healthy subjects Parameter Cmax (pg/mL) AUC0-48 h (pg·h/mL) AUC0-∞ (pg·h/mL) Tmax (h) T1/2 (h)
PCN
DM-PCN
339 ± 196 1127 ± 942 1270 ± 1062 1.1 ± 0.5 8.7 ± 7.3
536 ± 127 8732 ± 1593 9803 ± 1798 3.7 ± 2.3 14.3 ± 2.6
concentrations. Taking all these points into consideration, samples can be stored and prepared using routine laboratory conditions without special attention. Application to clinic pharmacokinetics study The described LC-MS/MS method was successfully applied to quantify the human plasma samples of PCN up to 48 h after a single oral dose administration of a 2 mg PCN tablet to 10 healthy subjects. Figure 3 shows the mean plasma concentration vs time profile of PCN and DM-PCN. The pharmacokinetics parameters are summarized in Table 2.
Conclusion A sensitive LC-MS/MS approach was developed and validated for simultaneous quantification of PCN and DM-PCN in human plasma (LLOQ for PCN and DM-PCN was 10 and 25 pg/mL, respectively). This approach showed superior accuracy and precision, and was proven to be appropriate for pharmacokinetic studies of PCN.
References [1] L.Y. Wang, Y. Wang, J.Q. Zheng, B.H. Zhong, H. Liu, S.J. Dong, J.X. Ruan, K.L. Liu. Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers. Acta Pharmacol. Sin. 2005, 26, 527. [2] Y. Liu, Y. Kou, M. Xue, Y. Xu, L. He, J. Ruan, K. Liu. Structural elucidation of in vivo metabolites of phencynonate and its analogue thiencynonate in rats by HPLC-ESI-MSn. Talanta. 2010, 82, 1200. [3] L.Y. Wang, J.Q. Zheng, Y. Wang, B.H. Zhong, J. X. Ruan, L.L. Liu. Comparative study on pharmacological effects of DM-phencynonate
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LC/MS quantifiaciton of phencynonate and N-demethyl phencynonate hydrochloride and its optical isomers. Acta Pharmacol. Sin. 2005, 26, 1187. [4] C. Liu, L. Yun. A new central anticholinergic anti-motion sickness drug phencynonate hydrochloride. Chin. J. Pharmacol. Toxic. 2005, 19, 311. [5] Y. Kou, Y. Liu, M. Xue, Y. Xu, H. Liu, J. Ruan, K. Liu. Comparative pharmacokinetics and distribution kinetics in brain of phencynonate enantiomers in rats. Int. J. Pharm. 2008, 353, 88. [6] Y. Kou, Y. Xu, M. Xue, J. Ruan, Z. Zhang, K. Liu. Liquid chromatographytandem mass spectrometry method for determination of phencynonate in rat blood and urine. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005, 828, 75.
Drug Testing and Analysis
[7] J. Li, Z. Zhang, J. Ruan, S. Wang, K. Liu. Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007, 855, 180. [8] Y. Xu, Y. Kou, M. Xue, Y. Liu, J. Ruan, Z. Zhang, K. Liu. Determination of thiencynonate by liquid chromatographic-mass spectrometry and its application to pharmacokinetics in rats. J. Pharm. Biomed. Anal. 2006, 42, 149. [9] US FDA. Guidance for Industry: Bioanalytical Method Validation. Available at: www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM368107.pdf [Sep 2013]
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Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/dta
Short communication Received: 3 March 2015
Revised: 17 April 2015
Accepted: 1 May 2015
Published online in Wiley Online Library: 20 May 2015
(www.drugtestinganalysis.com) DOI 10.1002/dta.1819
Simultaneous quantification of phencynonate and its active metabolite N-demethyl phencynonate in human plasma using liquid chromatography and isotope-dilution mass spectrometry Zhengang Chen, Hui Xie, Jinbo Liu and Guangshun Wang* A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously quantify phencynonate (PCN) and its major metabolite N-demethyl phencynonate (DM-PCN) in human plasma. Following one-step liquid-liquid extraction, the analytes were separated on a reversed-phase C18 column. Methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was used as isocratic mobile phase at a flow-rate of 0.3 mL/min. An API 5000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. Multiple reaction monitoring using the transition of m/z 358.4 → m/z 156.2, m/z 344.4 → m/z 142.2, and m/z 361.3 → m/z 159.2 was performed to quantify PCN, DM-PCN, and the internal standard (D3-PCN), respectively. This approach showed a lower limit of quantification of 10 pg/mL and 25 pg/mL for PCN and DM-PCN in plasma, respectively. This sensitivity was at least 50-fold superior to previously reported ones and thus enabled the approach well applicable to low-dose pharmacokinetic studies. The intra- and inter-day precisions were less than 14.2 % at each QC level for both PCN and DM-PCN. The inter-day relative errors ranged from -1.9% to -4.9% for PCN, and from 0.6% to 6.4% for DM-PCN. As a proof of principle, the validated method was successfully applied to simultaneous quantification of circulating PCN and DM-PCN in healthy subjects after a single oral administration of 2 mg phencynonate hydrochloride pellet. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: phencynonate; N-demethyl phencynonote; isotope-dilution LC-MS; clinic pharmacokinetics
Introduction
Experimental
Phencynonate (PCN, 3-Methyl-3-azabicyclo(3,3,1)nonanyl-9-α-yl-αcyclopentyl-α-phenyl-α-glycolate, Figure 1) is an anti-cholinergic agent showing therapeutic effects towards motion sickness, epilepsy and Parkinson’s disease.[1] PCN undergoes hepatic dealkylation and converts to its active metabolite N-demethyl phencynonate in vivo (DM-PCN, Figure 1).[2–4] The pre-clinical pharmacokinetics, distribution, and metabolic pathways of PCN in experimental animals have been well described. Additionally, several approaches were developed to quantify PCN or its analogues in the body fluids/tissues of experimental animals.[2,5–8] Kou et al. reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach to determine PCN enantiomers in rat blood with a low limit of quantification (LLOQ) of 1.0 ng/mL.[5] Li et al. reported an LC-MS/MS method to quantify PCN in rat plasma with an LLOQ of 0.5 ng/mL.[7] However, the human pharmacokinetics of PCN and its major metabolite DM-PCN have not been demonstrated to date. As the clinical dose of PCN is as low as 2 mg, a sensitive method is thus essential for the human pharmacokinetics study. In this work, a sensitive and selective LC-MS/MS method was developed and validated to quantify PCN and DM-PCN simultaneously in human plasma. This method was at least 50-fold more sensitive than previously reported ones and was well applicable to low-dose clinical pharmacokinetic studies.
Chemicals and reagents
LC–MS/MS The liquid chromatography system (Agilent, Santa Clara, CA, USA) was equipped with an LC-G1312A pump system and a G1367B autosampler. The chromatographic separation was achieved on an Agela C18 column (50 × 2.1 mm, 5 μm, Agela, Tianjin, China) at 25°C. The mobile phase was consisted of methanol and 0.02%
* Correspondence to: Guangshun Wang, Bao Di Clinical College of Tianjin Medical University, No.8 Guang Chuan Road, Baodi District, Tianjin, China. E-mail: [email protected] Bao Di Clinical College of Tianjin Medical University, Tianjin 301800, China
Copyright © 2015 John Wiley & Sons, Ltd.
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Drug Test. Analysis 2015, 7, 843–847
Phencynonate hydrochloride was provided by Beijin Hwells Pharmaceuticals (Beijing, China). D3-phencynonate (internal standard, IS) was purchased from High Standard Products Corporation (Westminster, CA, USA). DM-PCN was provided by Institute of Pharmacology & Toxicology of AMMS (Beijing, China). HPLC-grade acetonitrile and methanol were purchased from Dikma Technologies Inc. (Lake Forest, CA, USA). All other reagents were of analytical grade.
Drug Testing and Analysis
Z. Chen et al. Method validation
Figure 1. Chemical structures of (A) PCN, (B) DM-PCN and (C) D3-PCN (internal standard).
formic acid in 10 mM ammonium acetate (62:38, v/v) and run at a flow-rate of 0.3 mL/min. Each run time was 4 min. Mass spectrometric detection was performed on an API 5000 triple quadrupole instrument equipped with a TurboIonspray interface (AB SCIEX, Toronto, Canada). The data was acquired and processed using Analyst 1.5 software. Multiple reaction monitoring (MRM) analysis was applied in a positive ionization mode to detect precursor to product ion transitions at m/z 358.4 → m/z 156.2 with CE equal to 22 eV for PCN, m/z 344.4 →m/z 142.2 with CE equal to 30 eV for DM-PCN , and m/z 361.3 → m/z 159.2 with CE equal to 22 eV for IS. The ion spray voltage set at +5.5 kV and the heater gas temperature set to 600°C. The nebulizer gas (Gas 1) set at 0.36 MPa, a heater gas (Gas 2) of 0.31 MPa, a curtain gas of 0.069 MPa, and a collision gas of 0.055 MPa.
Sample preparation Once thawed, the plasma samples were vortexed and 200 μL plasma samples were taken. The samples were spiked with 25 μL of the working IS solution, 20 μL of an acetonitrile–water solution (50:50, v/v), 100 μL of methanol, and 20 μL of 20% ZnSO4 aqueous solution. The mixture was then vortex-mixed for 30 s and added to 1 mL of ethyl acetate. Finally, the mixture was vortexed for 30 s, twice. The two phases were separated through centrifugation at 3000 × g for 10 min at room temperature. The upper organic layer was transferred into another tube and completely evaporated under a stream of nitrogen. The residue was reconstituted with 200 μL of acetonitrile–water (50:50, v/v) and a 10-μL aliquot of the reconstituted extract was injected into LC–MS/MS system.
Standard, QC, and IS preparation
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Primary stock solutions of PCN at 0.994 mg/mL and DM-PCN at 0.519 mg/mL were prepared by dissolving accurately weighed amounts of the reference substances in acetonitrile–water (50:50, v/v). The stock solutions were serially diluted with acetonitrile– water (50:50, v/v) to give working solutions at the following concentrations for PCN and DM-PCN: 0.1/0.25, 0.2/0.5, 0.5/1.25, 1.25/3.13, 5/12.5, 12.5/31.3, 25/62.5, 40/100, and 50/125 ng/mL. Two other stock solutions were independently prepared and diluted with human plasma to achieve QC samples at concentrations of 30/75 pg/mL (LQC), 1500/3750 pg/mL (MQC), and 3500/8750 pg/mL (HQC) for PCN and DM-PCN. IS solution (100 ng/mL) was prepared by diluting the 8 mg/mL D3PCN stock solution with the acetonitrile–water mixture (50:50, v/v). All solutions were kept at 4°C and brought to room temperature before use. QC samples were kept at –70°C, brought to room temperature until completely thawed, and then vortexed before use. The calibration standards were prepared by spiking 200 μL blank plasma with 20 μL of the corresponding working solutions.
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The method was validated by verifying specificity, linearity, precision and accuracy, recovery, matrix effect, and stability. The method validation was performed according to FDA guidelines for Bioanalytical Method Validation.[9] Method specificity was evaluated by assaying human blank plasma samples from six different donors and LLOQ samples. LLOQ is defined as the lowest concentration of the analyte that is at least 5 times the response compared to blank response and that can be determined with acceptable precision and accuracy [six replicates in three validation days with a relative standard deviation (RSD) below 20% and a relative error (RE) within ±20%]. Linearity was assessed by plotting calibration curves in human plasma duplicated in three separate runs. The curves were fitted by a linear weighted (1/x2) least squares regression method through measurement of the peak-area ratios of the analytes to the IS solution. To evaluate the precision and accuracy of the method, QC samples at three concentration levels (30, 1500, and 3500 pg/mL for PCN, and 75, 3750, and 8750 pg/mL for DM-PCN) were analyzed in six replicates in five different batches within three validation days. The RSD determined assay precision, along with one-way analysis of variance. As well, the RSD sorted out sources of variance due to within- and between-run factors. Assay accuracy was expressed as the RE, or (observed concentration – nominal concentration) / (nominal concentration) × 100 %. The required accuracy was within ± 15 %, and the intra- and inter-day precision values were not to exceed 15 %. The recovery of the extraction procedure was estimated at three concentration levels (30, 1500, and 3500 pg/mL for PCN, and 75, 3750, and 8750 pg/mL for DM-PCN) by comparing the peak areas of blank samples spiked before and after liquid-liquid extraction (LLE). Spike-after-extraction samples represented 100% recovery. Matrix effects were assessed to determine whether potential ion suppression or enhancement due to the co-elution of matrix components existed in the experiment. The corresponding peak areas of the analytes from the spike-after-extraction samples at low, medium, and high concentration levels were compared to the standard solution at the same concentration in the mobile phase. The variability in the values, expressed as RSD (%), was a measure of the relative matrix effect for the analyte, which should be less than 15%. The stability of PCN and DM-PCN in human plasma was evaluated by analyzing replicates (n = 3) of plasma samples exposed to different conditions (time and temperature) at 30, 1500, 3500 pg/mL for PCN, and 75, 375, and 875 pg/mL for DM-PCN. These results were compared to those obtained from freshly prepared plasma samples. The analytes were considered to be stable in the biological matrix when 85–115 % of the initial concentration was retained.
Clinical application The developed method was applied to determine the plasma concentrations of PCN and DM-PCN. The study was approved by the Ethics Committee of Baodi Hospital. A total of 10 healthy male Chinese volunteers were included in the study. Blood samples were drawn, through a sodium citrate anticoagulative tube, at the baseline (before drug administration) and at 0.25, 0.50, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10, 12, 24, 36, and 48 h after oral administration of 2 mg PCN tablet. Plasma samples were obtained by centrifugation at 2000 × g for 10 min and frozen at –70°C until analysis.
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Drug Test. Analysis 2015, 7, 843–847
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LC/MS quantifiaciton of phencynonate and N-demethyl phencynonate Time profiles of plasma concentrations of PCN and DM-PCN were acquired for each subject. The major non-compartmental pharmacokinetic parameters of PCN and DM-PCN were then calculated. The maximum plasma concentration (Cmax) and the time of occurrence (Tmax) were obtained directly from the measured data. The terminal elimination rate constant (ke) was estimated by linear least-squares regression of the terminal portion of the plasma concentration–time curve, and the corresponding elimination half-life (T1/2) was then calculated as 0.693/ke. The area under the plasma concentration-time curve (AUC) was calculated according to the linear trapezoidal rule to the last measurable point (AUC0 t) or to infinity (AUC0 ∞) by AUC0 t + Ct/ke, where Ct was the last measurable drug concentration.
bond. Accordingly, an MRM transition channel of m/z 358.4 → m/z 156.2 was chosen to quantify PCN. A transition of m/z 344.4 → m/z 142.2 was chosen for DM-PCN. Deuterated PCN (D3-PCN) was chosen as internal standard to further improve the assay accuracy and precision. The corresponding transition of IS was set as m/z 361.3 → m/z 159.2. A variety of mobile phase solvents and additives were investigated and evaluated in terms of both sensitivity and separation efficiency. An isocratic elution using methanol and 0.02% formic acid in 10 mM ammonium acetate (62:38, v/v) was finally employed. The optimal flow rate was set at 0.3 mL/min. No appreciable interferences from plasma were observed at the retention times of targeted analytes (Figure 2). Method validation Linearity of calibration standards
Results and discussion Optimization of mass spectrometric and chromatographic conditions Low dose PCN (e.g. 2 mg/day) substantially attenuates the adverse effects of anti-cholinergics in clinic applications, albeit poses a daunting challenge for pharmacokinetic study. To achieve the necessary sensitivity (pg/mL level), the LC-MS conditions were systemically optimized as described below. Electrospray ionization (ESI) at positive ionization mode was selected for the quantification of PCN and DM-PCN. Under the experiment conditions, soft ionization in the ESI source produced the protonated molecules [M + H]+ of PCN and DM-PCN at m/z 358 and m/z 344 as the base peak, respectively. The product ion mass spectrum of [M + H]+ of PCN showed a major fragment at m/z 156 resulting from the cleavage of the ester
Plotted calibration curves and correlation coefficients > 0.99 confirm that the calibration curves were linear over the concentration range 10 5000 pg/mL and 25 12500 pg/mL for PCN and DM-PCN, respectively. Typical curves for PCN and DM-PCN are as follows: y = 3.44 × 10 3 x 1.71 × 10 3, r = 0.9963, and y = 1.10 × 10 3 x 6.26×10 4, r = 0.9979, where y represents the ratio of the analyte peak area to that of IS and x is the plasma concentration of the analyte. Assay specificity and LLOQ Three typical MRM chromatograms from the study of PCN and DM-PCN in human plasma are shown in Figure 2. No interfering peaks were observed in blank plasma (Figure 2A). A representative MRM chromatogram of blank plasma spiked with PCN (10 pg/mL),
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Figure 2. Representative chromatograms for PCN (I), DM-PCN (II), and D3-PCN (IS, III) in human plasma samples. (A) the MRM chromatograms of the blank plasma sample; (B) the MRM chromatograms of blank plasma spiked with PCN (10 pg/mL), DM-PCN (25 pg/mL), and D3-PCN (500 pg/mL); (C) the MRM chromatograms of a plasma sample taken from a volunteer 5 h after oral administration of 2 mg PCN.
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Z. Chen et al.
DM-PCN (25 pg/mL) and IS (500 pg/mL) is shown in Figure 2B. A sample from a volunteer 0.5 h after oral administration of 2 mg PCN is shown in Figure 2C. For PCN, DM-PCN, and IS, the chromatograms were free from endogenous matrix interference at their respective retention times. For PCN and DM-PCN, the LC-MS/MS method yielded an LLOQ of 10 pg/mL and 25 pg/mL with accuracies of –1.32% and 2.5% in terms of RE; the precision values were less than 3.9% and 10.4% in terms of RSD, respectively. The data are shown in Table 1. The signal-to-noise ratio (S/N) of the LLOQ was 22.5 for PCN and 12.8 for DM-PCN, respectively (as shown in Figure 2B). Precision and accuracy The intra- and inter-day precision and accuracy were calculated by analysis of variance based on replicate analyses (n = 6) of QC samples. In this study, the intra- and inter-day precision values were less than 12.6% and 14.2% for each QC level of PCN and DM-PCN, respectively. The inter-day relative errors were –1.9%, –2.9%, and –4.9%, and 6.0%, 6.4%, and 0.6% for low, medium, and high concentrations of PCN and DM-PCN, respectively. These data indicate reproducible LC-MS/MS results and prove that the assay was accurate and reliable. The accuracy and precision data are shown in Table 1. Recovery The extraction recovery of PCN determined at concentrations of 30, 1500, and 3500 pg/mL were 93.3%, 105%, and 107%, respectively. The extraction recovery of DM-PCN determined at concentrations of 75, 3750, and 8750 pg/mL were 72.5%, 79.5%, 84.1%, respectively. The extraction recovery of IS was determined to be 99.4%. Matrix effect The matrix factors of PCN determined at concentrations of 30, 1500, and 3500 pg/mL were 115%, 104%, and 102%, respectively, and the RSD values from six lots of plasma were less than 4.7%. The matrix factors of PCN determined at concentrations of 75, 3750, and 8750 pg/mL were 111%, 99.9%, and 93.6%, respectively, and the RSD values from six lots of plasma were less than 4.5%. Stability For both PCN and DM-PCN, the plasma samples were stable for 6 h at 25°C, 7 h after preparation at 25°C, 53 h after preparation at 4°C and after 4 freeze-thaw cycles ( 70 to 25°C) on consecutive days; the RE values were within ±15 % for all low, media, and high Table 1. Accuracy and precision for the analysis of PCN and DM-PCN in human plasma Compound
PCN
DM-PCN
Nominal Mean Relative Intra-day Inter-day plasma measured error RSD RSD concentration concentration
846
(pg/mL)
(pg/mL)
10 30 1500 3500 25 75 3750 8750
9.87 29.4 1457 3329 24.7 79.5 3992 8805
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(%) 1.3 1.9 2.9 4.9 2.5 6.0 6.4 0.6
(%)
(%)
3.9 6.6 2.7 4.0 10.4 7.0 5.9 8.9
12.6 11.0 12.2 13.6 12.1 14.2
Figure 3. Mean plasma concentration-time curve of PCN and DM-PCN in healthy subjects (n = 10, mean ± SD).
Table 2. The main pharmacokinetic parameters of PCN and DM-PCN after an oral administration of 2 mg PCN hydrochloride to ten healthy subjects Parameter Cmax (pg/mL) AUC0-48 h (pg·h/mL) AUC0-∞ (pg·h/mL) Tmax (h) T1/2 (h)
PCN
DM-PCN
339 ± 196 1127 ± 942 1270 ± 1062 1.1 ± 0.5 8.7 ± 7.3
536 ± 127 8732 ± 1593 9803 ± 1798 3.7 ± 2.3 14.3 ± 2.6
concentrations. Taking all these points into consideration, samples can be stored and prepared using routine laboratory conditions without special attention. Application to clinic pharmacokinetics study The described LC-MS/MS method was successfully applied to quantify the human plasma samples of PCN up to 48 h after a single oral dose administration of a 2 mg PCN tablet to 10 healthy subjects. Figure 3 shows the mean plasma concentration vs time profile of PCN and DM-PCN. The pharmacokinetics parameters are summarized in Table 2.
Conclusion A sensitive LC-MS/MS approach was developed and validated for simultaneous quantification of PCN and DM-PCN in human plasma (LLOQ for PCN and DM-PCN was 10 and 25 pg/mL, respectively). This approach showed superior accuracy and precision, and was proven to be appropriate for pharmacokinetic studies of PCN.
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LC/MS quantifiaciton of phencynonate and N-demethyl phencynonate hydrochloride and its optical isomers. Acta Pharmacol. Sin. 2005, 26, 1187. [4] C. Liu, L. Yun. A new central anticholinergic anti-motion sickness drug phencynonate hydrochloride. Chin. J. Pharmacol. Toxic. 2005, 19, 311. [5] Y. Kou, Y. Liu, M. Xue, Y. Xu, H. Liu, J. Ruan, K. Liu. Comparative pharmacokinetics and distribution kinetics in brain of phencynonate enantiomers in rats. Int. J. Pharm. 2008, 353, 88. [6] Y. Kou, Y. Xu, M. Xue, J. Ruan, Z. Zhang, K. Liu. Liquid chromatographytandem mass spectrometry method for determination of phencynonate in rat blood and urine. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005, 828, 75.
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