http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2014; 44(8): 743–748 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.887802
RESEARCH ARTICLE
Gradient elution liquid chromatography mass spectrometry determination of acetylcorynoline in rat plasma and its application to a pharmacokinetic study Congcong Wen1, Jinzhang Cai2, Chongliang Lin3, Jianshe Ma4, and Xianqin Wang4
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
1
Laboratory Animal Centre, Wenzhou Medical University, Wenzhou, China, 2The Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, China, 3The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, China, and 4Analytical and Testing Center, Wenzhou Medical University, Wenzhou, China Abstract
Keywords
1. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. A sensitive and selective liquid chromatography mass spectrometry method for determination of acetylcorynoline in rat plasma was developed over the range of 5–1000 ng/mL to characterize the pharmacokinetic properties. 2. Chromatographic separation was achieved on a C18 (2.1 mm 150 mm, 5 mm) 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 carbamazepine as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) 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 using target ions m/z 410 for acetylcorynoline and m/z 237 for the IS. 3. Mean recoveries of acetylcorynoline in rat plasma were in the range of 72.3–87.6%. Matrix effects for acetylcorynoline were measured to be between 88.7% and 93.5%. Coefficient of variation of intra-day and inter-day precision were both 513%. The accuracy of the method ranged from 95.8% to 112.1%. The analyte was stable under auto-sampler, room temperature, freeze-thaw and long-term (20 days), the bias in concentration was within ±15% of their nominal values. 4. The LC-MS method for the determination of acetylcorynoline in rat plasma utilizing 100 mL of plasma with an LLOQ of 5.0 ng/mL developed and validated, it was sensitive, selective and simple. This method was successfully applied in pharmacokinetic study of acetylcorynoline after intravenous administration of single dosage 3 mg/kg in rats.
Acetylcorynoline, LC-MS, pharmacokinetics, rat plasma
Introduction Corydalis bungeana Turcz. (Papaveraceae) is a perennial herb distributed in many parts of the World, such as the northern and eastern parts of China and the northern part of the Korean peninsula, it has been listed for treatments such as upper respiratory tract infections, tonsillitis, influenza, bronchitis, pyelonephritis and acute nephritis (Xie et al., 2004). Acetylcorynoline (Figure 1) is the major alkaloid component derived from C. bungeana herbs. Pharmacological research showed that acetylcorynoline has sedative, hypnotic and bacteriostastic activity (Wang et al., 2012). Oral administration of acetylcorynoline has been shown to significantly decrease elevated serum levels of glutamate pyruvate
Address for correspondence: Xianqin Wang, Analytical and Testing Center, Wenzhou Medical University, University-Town, Wenzhou 325035, China. Tel: +86 577 86699156. Fax: +86 577 86699156. E-mail: [email protected]
History Received 13 December 2013 Revised 21 January 2014 Accepted 22 January 2014 Published online 10 February 2014
transaminase and liver damage induced by injection of carbon tetrachloride, acetaminophen or thioacetamide in mice (Wei & Liu, 1997). Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of dendritic cells maturation and function (Fu et al., 2013b). Acetylcorynoline significantly decreases dopaminergic neuron degeneration induced by 6-hydroxydopamine in BZ555 (Pdat-1:GFP; green fluorescent protein [GFP] visible in dopaminergic neurons) strain; prevents a-synuclein aggregation; recovers lipid content in OW13 (Punc-54:a-synuclein: YFP + unc-119; human a-synuclein protein with yellow fluorescent protein [YFP] observable in the muscles) strain; restores food-sensing behavior, and dopamine levels; and prolongs life-span in 6-hydroxydopamine-treated nicotinic muscle receptor strain, thus showing its potential as a possible anti-Parkinsonian drug (Fu et al., 2013a). Until now, to our best knowledge, there is no bioanalytical method for determination of acetylcorynoline to characterize the pharmacokinetic properties. Liquid chromatography in combination with mass spectrometry (LC-MS) is a
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
744
C. Wen et al.
Figure 1. Chemical structure of acetylcorynoline (a) and carbamazepine (IS, b).
well-established analytical tool in many fields of application (Ma et al., 2012; Wang et al., 2011; Zhou et al., 2013a,b). Herein, we report the analytical method for the quantitation of acetylcorynoline in rat plasma in support of a project to understand pharmacology features. This method is accurate, reliable, sensitive and simple and can be used to analyze varied amounts of acetylcorynoline in rat plasma for phamacokinetic study.
Experimental Chemicals and reagents Acetylcorynoline (purity >98%) was purchased from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Carbamazepine (IS, purity >98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA). Instrumentation and conditions Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatography (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 150 mm, 5 mm) 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 program was conducted for chromatographic separation with mobile phase
Xenobiotica, 2014; 44(8): 743–748
Figure 2. Mass spectrum of acetylcorynoline (a) and carbamazepine (IS, b).
A (0.1% formic acid), and mobile phase B (acetonitrile) as follows: 0–1.0 min (10–10% B), 1.0–4.0 min (10–80% B), 4.0–7.0 min (80–80% B), 7.0–8.0 min (80–10% B) and 8.0–12.0 min (10–10% B). Drying gas flow and nebuliser pressure were set at 7 L/min and 25 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 C and 3500 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 410 for acetylcorynoline (Figure 2a) and m/z 237 for carbamazepine (IS, Figure 2b), in positive ion electrospray ionization interface. Calibration standards and quality control samples The stock solutions of acetylcorynoline (1.0 mg/mL) and carbamazepine (IS, 1.0 mg/mL) were prepared in methanol, respectively. Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 2.0-mg/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. Acetylcorynoline calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 5–1000 ng/mL for acetylcorynoline with IS (200 ng/mL) in rat plasma, prepared at concentrations of 5, 10, 20, 50, 100, 200, 500 and 1000 ng/mL, each by adding 10 mL of the appropriate working solution to 190 mL of blank rat plasma, followed by short vortex mixing. Quality control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations
DOI: 10.3109/00498254.2014.887802
(10, 400 and 800 ng/mL). The analytical standards and QC samples were stored at 20 C. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5-mL centrifuge tube, an aliquot of 10 mL of the internal standard working solution (2.0 mg/mL) was added to 100 mL of collected plasma sample followed by the addition of 200 mL acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14 900 g for 10 min, the supernatant (2 mL) was injected into the LC–ESI–MS system for analysis.
LC-MS determination of acetylcorynoline in rat plasma
745
to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the readyto-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The freeze/thaw stability was evaluated after three complete freeze/thaw cycles (20 to 25 C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at 20 C for 20 days. The stability of the IS (200 ng/mL) was evaluated in a similar way. Pharmacokinetic study
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
Method validation The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA, 2001) and European Medicines Agency (EMA, 2011) for validation of bioanalytical methods. 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 six blank rat plasma, blank plasma spiked acetylcorynoline and IS and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of acetylcorynoline 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 5– 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 (520%). Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (10, 400 and 800 ng/mL) in three validation days. The precision was expressed by coefficient of variation (CV). The recovery of acetylcorynoline (A/B 100%) was evaluated by comparing peak area ratios of QC samples (A) with those of reference QC solutions reconstituted in blank plasma protein precipitation (B, n ¼ 6). The recover of the IS was determined in a similar way. To evaluate the matrix effect (B/C 100%), blank rat plasma after protein precipitation and then spiked with the analyte at 10, 100 400 and 800 ng/mL (B). The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations (C), and this peak area ratio is defined as the matrix effect. The matrix effect, of IS was evaluated at the working concentration (200 ng/mL) in the same manner. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ) and the response was checked (Williams et al., 2012). The stabilities of acetylcorynoline in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 10 and 800 ng/mL, which were exposed
Male Sprague–Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of acetylcorynoline. All six rats were housed at 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, 0.75, 1, 1.5, 2, 3, 4, 6, 8 h after intravenous administration of acetylcorynoline (3 mg/kg). The blank blood samples (0.3 mL) were collected before administration. The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 mL) was stored at 20 C until analysis. Plasma acetylcorynoline concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
Results and discussion Method development Acetylcorynoline was a weak alkaline substances, and we chose the positive ionization interface in our work. Electrospray ionization (ESI+) source exhibited more sensitivity and better reproducibility for acetylcorynoline compared with an atmospheric pressure chemical ionization (APCI+) interface, and ESI+ was used in this work. The experiment used methanol 0.1% formic acid as mobile phase at first, but it produced high pressure, poor stability, long equilibrium time, so the selection of the organic phase was acetonitrile in our work. Mobile phase 0.1% aqueous formic acid was in purpose of increasing the degree of ionization ESI in positive ion source, and it improved sensitivity of acetylcorynoline. Liquid chromatography system using a gradient elution method could elute more residual impurities from column for each sample; furthermore it could reduce the damage of the biological sample to the column. The experiment was tried 10, 5 and 2 mL of injection volume. The 10-mL injection volumes would produce a certain matrix effects, it could reduce matrix effects with the reduction of injection volume, the result of matrix effects could be satisfied when 2 mL volumes was injected into LC-MS.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
746
C. Wen et al.
When plasma sample processing method used diethyl ether, chloroform, methylene chloride or ethyl acetate for liquid–liquid extraction, it needs drying and reconstitution, the process is relatively cumbersome. In this study, treatment of plasma by protein precipitation is simple and it does not require reconstitution and drying process, although it will dilute the concentration of acetylcorynoline in plasma, but with the use of liquid chromatography mass spectrometry could meet the requirements of pharmacokinetic study. The solvent for precipitation method, methanol, acetonitrile and methanol–acetonitrile (10/90, v/v), the result found that acetonitrile was more effective than methanol. When acetonitrile added with 10% methanol, plasma could be completely wash off from the centrifuge tube, and it provided higher recovery, so that the present study used a methanol–acetonitrile (10/90, v/v) as a protein precipitant. Select carbamazepine as IS, because its chromatographic performance was similar with acetylcorynoline, and they had similar retention time; they were all weak alkaline substances, and the recoveries of them were similar, both suitable for detection in positive ion electrospray ionization interface.
Xenobiotica, 2014; 44(8): 743–748
Selectivity and matrix effect Figure 3 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with acetylcorynoline and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The matrix effect for acetylcorynoline at concentrations of 10, 400 and 800 ng/mL were measured to be 90.3 ± 9.7%, 93.5 ± 5.1% and 88.7 ± 3.1% (n ¼ 6), respectively. The matrix effect for IS (200 ng/mL) was measured to be 102.1 ± 3.7% (n ¼ 6). As a result, matrix effect from plasma was negligible in this method. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 5–1000 ng/mL for acetylcorynoline in rat plasma. Typical equation of the calibration curve was: y ¼ (0.00238 ± 0.00023) x + (0.02592 ± 0.02226), r ¼ 0.9971 ± 0.0016, where y represents the ratios of acetylcorynoline peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of acetylcorynoline in plasma was 5 ng/mL.
Figure 3. Representative LC-MS chromatograms of acetylcorynoline (1) and carbamazepine (IS, 2), (a) blank plasma; (b) blank plasma spiked with acetylcorynoline (LLOQ, 5 ng/mL) and IS (200 ng/mL), (c) a rat plasma sample 3 h after intravenous administration of single dosage 3 mg/kg acetylcorynoline (55 ng/mL).
LC-MS determination of acetylcorynoline in rat plasma
DOI: 10.3109/00498254.2014.887802
747
Table 1. Precision and accuracy for acetylcorynoline of QC sample in rat plasma (n¼6). Concentration (ng/mL) 5 10 400 800
CV (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
11.0 7.4 8.0 9.1
12.4 5.0 5.2 6.3
108.3 95.8 96.7 102.3
112.1 108.6 102.5 96.7
The precision and accuracy at LLOQ were 11.0% and 108.3%, respectively. The LOD, defined as a signal/noise ratio of 3, was 1.5 ng/mL for acetylcorynoline in rat plasma.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
Precision, accuracy and recovery The precision of the method was determined by calculating CV for QCs at three concentration levels over three validation days. Intra-day precision was 12% or less and the inter-day precision was 13% or less at each QC level. The accuracy of the method ranged from 95.8% to 112.1% at each QC level. Assay performance data are presented in Table 1. The results demonstrate that the values are within the acceptable range and the method is accurate and precise. Mean recoveries of acetylcorynoline at concentrations of 10, 400 and 800 ng/mL were measured to be 76.1 ± 7.4, 72.3 ± 6.4 and 87.6 ± 5.9% (n ¼ 6), respectively. The differences in the observed recovery rates at low–medium (72.3–76.1%) and high concentrations (87.6%), it may be an impact of the matrix, however the results of recovery could be acceptable. The recovery of the IS (200 ng/mL) was measured to be 93.4 ± 4.6%.
Figure 4. Mean plasma concentration time profile after intravenous administration of single dosage 3 mg/kg acetylcorynoline in six rats.
Carry-over
decreased very quickly, and the t1/2 was 1.8 h. The pharmacokinetic profile of acetylcorynoline in rat was characterized for the first time. It helps to better understand pharmacology features of acetylcorynoline.
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 four extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over. Stability The auto-sampler, room temperature, freeze–thaw and longterm (20 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of their nominal values, and the established method was suitable for the pharmacokinetic study. Application to pharmacokinetics The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after intravenous administration of a single 3 mg/kg dose of acetylcorynoline was shown in Figure 4. The area under the plasma concentration–time curve (AUC), the mean residence time (MRT), the plasma clearance (CL), apparent volume of distribution (V), the maximum plasma concentration (Cmax) and the halflife (t1/2) were estimated using non-compartmental calculations performed with DAS (Drug and statistics) software. The main pharmacokinetic parameters were summarized in Table 2. In vivo, the acetylcorynoline concentration was
Table 2. The main pharmacokinetic parameters after intravenous administration of single dosage 3 mg/kg acetylcorynoline in six rats. Parameters
Unit
Mean (±SD)
AUC(0–t) AUC(0–1) MRT(0–t) MRT(0–1) t1/2 CL V Cmax
ng/mL h ng/mL h H H H L/h/kg L/kg ng/mL
426.2 ± 94.2 441.8 ± 98.4 1.7 ± 0.2 2.0 ± 0.2 1.8 ± 0.1 7.3 ± 1.5 19.4 ± 4.2 550.6 ± 21.6
Conclusion The LC-MS method for determination of acetylcorynoline in rat plasma utilizing 100 mL of plasma with an LLOQ of 5.0 ng/mL developed and validated, it was sensitive, selective and simple. A time-saving protein precipitation procedure made the method readily applicable in a further clinical study. The LC-MS method successfully applied to a pharmacokinetic study of acetylcorynoline after intravenous administration of single dosage 3 mg/kg to rats. The pharmacokinetic profile of acetylcorynoline in rat was characterized for the first time.
Declaration of interest The authors declare no conflict of interest.
References European Medicines Agency (EMA). (2011). Guideline on bioanalytical method validation. Available from: http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/ 08/WC500109686.pdf [last accessed 7 Feb 2014]. Fu RH, Wang YC, Chen CS, et al. (2013a). Acetylcorynoline attenuates dopaminergic neuron degeneration and alpha-synuclein aggregation in
748
C. Wen et al.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
animal models of Parkinson’s disease. Neuropharmacology. [Epub ahead of print]. doi:10.1016/j.neuropharm.2013.08.007. Fu RH, Wang YC, Liu SP, et al. (2013b). Acetylcorynoline impairs the maturation of mouse bone marrow-derived dendritic cells via suppression of IkappaB kinase and mitogen-activated protein kinase activities. PloS One 8:e58398. Ma J, Dong J, Lin G, et al. (2012). Determination of curdione in rabbit plasma by liquid chromatography mass spectrometry. Biomed Chromatogr 26:655–9. US Food and Drug Administration. (2001). Guidance for industry: bioanalytical method validation. Available from: http://www.fda.gov/ downloads/Drugs/Guidances/ucm070107.pdf [last accessed 7 Feb 2014]. Wang X, Dong H, Shu X, et al. (2012). Large-scale separation of alkaloids from Corydalis bungeana Turcz. by pH-zone-refining counter-current chromatography. Molecules 17:14968–74. Wang X, Xiang Z, Cai X, et al. (2011). Determination of pethidine in human plasma by LC-MS/MS. Biomed Chromatogr 25:833–7.
Xenobiotica, 2014; 44(8): 743–748
Wei HL, Liu GT. (1997). Protective action of corynoline, acetylcorynoline and protopine against experimental liver injury in mice. Acta Pharma Sin 32:331–6. Williams JS, Donahue SH, Gao H, Brummel CL. (2012). Universal LCMS method for minimized carryover in a discovery bioanalytical setting. Bioanalysis 4:1025–37. Xie C, Veitch NC, Houghton PJ, Simmonds MS. (2004). Flavonoid glycosides and isoquinolinone alkaloids from Corydalis bungeana. Phytochemistry 65:3041–7. Zhou Y, Li J, He X, et al. (2013a). Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous determination of amlodipine, atorvastatin and its metabolites ortho-hydroxy atorvastatin and para-hydroxy atorvastatin in human plasma and its application in a bioequivalence study. J Pharm Biomed Anal 83C:101–7. Zhou Z, Wu X, Wei Q, et al. (2013b). Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma. Anal Bioanal Chem 405:6323–35.
RESEARCH ARTICLE
Gradient elution liquid chromatography mass spectrometry determination of acetylcorynoline in rat plasma and its application to a pharmacokinetic study Congcong Wen1, Jinzhang Cai2, Chongliang Lin3, Jianshe Ma4, and Xianqin Wang4
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
1
Laboratory Animal Centre, Wenzhou Medical University, Wenzhou, China, 2The Second Affiliated Hospital & Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou, China, 3The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, China, and 4Analytical and Testing Center, Wenzhou Medical University, Wenzhou, China Abstract
Keywords
1. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. A sensitive and selective liquid chromatography mass spectrometry method for determination of acetylcorynoline in rat plasma was developed over the range of 5–1000 ng/mL to characterize the pharmacokinetic properties. 2. Chromatographic separation was achieved on a C18 (2.1 mm 150 mm, 5 mm) 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 carbamazepine as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) 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 using target ions m/z 410 for acetylcorynoline and m/z 237 for the IS. 3. Mean recoveries of acetylcorynoline in rat plasma were in the range of 72.3–87.6%. Matrix effects for acetylcorynoline were measured to be between 88.7% and 93.5%. Coefficient of variation of intra-day and inter-day precision were both 513%. The accuracy of the method ranged from 95.8% to 112.1%. The analyte was stable under auto-sampler, room temperature, freeze-thaw and long-term (20 days), the bias in concentration was within ±15% of their nominal values. 4. The LC-MS method for the determination of acetylcorynoline in rat plasma utilizing 100 mL of plasma with an LLOQ of 5.0 ng/mL developed and validated, it was sensitive, selective and simple. This method was successfully applied in pharmacokinetic study of acetylcorynoline after intravenous administration of single dosage 3 mg/kg in rats.
Acetylcorynoline, LC-MS, pharmacokinetics, rat plasma
Introduction Corydalis bungeana Turcz. (Papaveraceae) is a perennial herb distributed in many parts of the World, such as the northern and eastern parts of China and the northern part of the Korean peninsula, it has been listed for treatments such as upper respiratory tract infections, tonsillitis, influenza, bronchitis, pyelonephritis and acute nephritis (Xie et al., 2004). Acetylcorynoline (Figure 1) is the major alkaloid component derived from C. bungeana herbs. Pharmacological research showed that acetylcorynoline has sedative, hypnotic and bacteriostastic activity (Wang et al., 2012). Oral administration of acetylcorynoline has been shown to significantly decrease elevated serum levels of glutamate pyruvate
Address for correspondence: Xianqin Wang, Analytical and Testing Center, Wenzhou Medical University, University-Town, Wenzhou 325035, China. Tel: +86 577 86699156. Fax: +86 577 86699156. E-mail: [email protected]
History Received 13 December 2013 Revised 21 January 2014 Accepted 22 January 2014 Published online 10 February 2014
transaminase and liver damage induced by injection of carbon tetrachloride, acetaminophen or thioacetamide in mice (Wei & Liu, 1997). Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of dendritic cells maturation and function (Fu et al., 2013b). Acetylcorynoline significantly decreases dopaminergic neuron degeneration induced by 6-hydroxydopamine in BZ555 (Pdat-1:GFP; green fluorescent protein [GFP] visible in dopaminergic neurons) strain; prevents a-synuclein aggregation; recovers lipid content in OW13 (Punc-54:a-synuclein: YFP + unc-119; human a-synuclein protein with yellow fluorescent protein [YFP] observable in the muscles) strain; restores food-sensing behavior, and dopamine levels; and prolongs life-span in 6-hydroxydopamine-treated nicotinic muscle receptor strain, thus showing its potential as a possible anti-Parkinsonian drug (Fu et al., 2013a). Until now, to our best knowledge, there is no bioanalytical method for determination of acetylcorynoline to characterize the pharmacokinetic properties. Liquid chromatography in combination with mass spectrometry (LC-MS) is a
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
744
C. Wen et al.
Figure 1. Chemical structure of acetylcorynoline (a) and carbamazepine (IS, b).
well-established analytical tool in many fields of application (Ma et al., 2012; Wang et al., 2011; Zhou et al., 2013a,b). Herein, we report the analytical method for the quantitation of acetylcorynoline in rat plasma in support of a project to understand pharmacology features. This method is accurate, reliable, sensitive and simple and can be used to analyze varied amounts of acetylcorynoline in rat plasma for phamacokinetic study.
Experimental Chemicals and reagents Acetylcorynoline (purity >98%) was purchased from the Chengdu Mansite Pharmaceutical CO. LTD. (Chengdu, China). Carbamazepine (IS, purity >98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA). Instrumentation and conditions Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with a 1200 Series liquid chromatography (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 150 mm, 5 mm) 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 program was conducted for chromatographic separation with mobile phase
Xenobiotica, 2014; 44(8): 743–748
Figure 2. Mass spectrum of acetylcorynoline (a) and carbamazepine (IS, b).
A (0.1% formic acid), and mobile phase B (acetonitrile) as follows: 0–1.0 min (10–10% B), 1.0–4.0 min (10–80% B), 4.0–7.0 min (80–80% B), 7.0–8.0 min (80–10% B) and 8.0–12.0 min (10–10% B). Drying gas flow and nebuliser pressure were set at 7 L/min and 25 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 C and 3500 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 410 for acetylcorynoline (Figure 2a) and m/z 237 for carbamazepine (IS, Figure 2b), in positive ion electrospray ionization interface. Calibration standards and quality control samples The stock solutions of acetylcorynoline (1.0 mg/mL) and carbamazepine (IS, 1.0 mg/mL) were prepared in methanol, respectively. Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 2.0-mg/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. Acetylcorynoline calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 5–1000 ng/mL for acetylcorynoline with IS (200 ng/mL) in rat plasma, prepared at concentrations of 5, 10, 20, 50, 100, 200, 500 and 1000 ng/mL, each by adding 10 mL of the appropriate working solution to 190 mL of blank rat plasma, followed by short vortex mixing. Quality control (QC) samples were prepared by the same way as the calibration standards, three different plasma concentrations
DOI: 10.3109/00498254.2014.887802
(10, 400 and 800 ng/mL). The analytical standards and QC samples were stored at 20 C. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5-mL centrifuge tube, an aliquot of 10 mL of the internal standard working solution (2.0 mg/mL) was added to 100 mL of collected plasma sample followed by the addition of 200 mL acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14 900 g for 10 min, the supernatant (2 mL) was injected into the LC–ESI–MS system for analysis.
LC-MS determination of acetylcorynoline in rat plasma
745
to different conditions. These results were compared with those obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spiked samples at room temperature for 2 h, and the readyto-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. The freeze/thaw stability was evaluated after three complete freeze/thaw cycles (20 to 25 C) on consecutive days. The long-term stability was assessed after storage of the standard spiked plasma samples at 20 C for 20 days. The stability of the IS (200 ng/mL) was evaluated in a similar way. Pharmacokinetic study
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
Method validation The method was validated for selectivity, linearity, accuracy, precision, recovery and stability according to the guidelines set by the United States Food and Drug Administration (FDA, 2001) and European Medicines Agency (EMA, 2011) for validation of bioanalytical methods. 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 six blank rat plasma, blank plasma spiked acetylcorynoline and IS and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of acetylcorynoline 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 5– 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 (520%). Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (10, 400 and 800 ng/mL) in three validation days. The precision was expressed by coefficient of variation (CV). The recovery of acetylcorynoline (A/B 100%) was evaluated by comparing peak area ratios of QC samples (A) with those of reference QC solutions reconstituted in blank plasma protein precipitation (B, n ¼ 6). The recover of the IS was determined in a similar way. To evaluate the matrix effect (B/C 100%), blank rat plasma after protein precipitation and then spiked with the analyte at 10, 100 400 and 800 ng/mL (B). The corresponding peak areas were then compared to those of neat standard solutions at equivalent concentrations (C), and this peak area ratio is defined as the matrix effect. The matrix effect, of IS was evaluated at the working concentration (200 ng/mL) in the same manner. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ) and the response was checked (Williams et al., 2012). The stabilities of acetylcorynoline in rat plasma were evaluated by analyzing three replicates of plasma samples at the concentrations of 10 and 800 ng/mL, which were exposed
Male Sprague–Dawley rats (200–220 g) were obtained from Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) used to study the pharmacokinetics of acetylcorynoline. All six rats were housed at 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, 0.75, 1, 1.5, 2, 3, 4, 6, 8 h after intravenous administration of acetylcorynoline (3 mg/kg). The blank blood samples (0.3 mL) were collected before administration. The samples were immediately centrifuged at 3000 g for 10 min. The plasma obtained (100 mL) was stored at 20 C until analysis. Plasma acetylcorynoline concentration versus time data for each rat was analyzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou Medical University, China).
Results and discussion Method development Acetylcorynoline was a weak alkaline substances, and we chose the positive ionization interface in our work. Electrospray ionization (ESI+) source exhibited more sensitivity and better reproducibility for acetylcorynoline compared with an atmospheric pressure chemical ionization (APCI+) interface, and ESI+ was used in this work. The experiment used methanol 0.1% formic acid as mobile phase at first, but it produced high pressure, poor stability, long equilibrium time, so the selection of the organic phase was acetonitrile in our work. Mobile phase 0.1% aqueous formic acid was in purpose of increasing the degree of ionization ESI in positive ion source, and it improved sensitivity of acetylcorynoline. Liquid chromatography system using a gradient elution method could elute more residual impurities from column for each sample; furthermore it could reduce the damage of the biological sample to the column. The experiment was tried 10, 5 and 2 mL of injection volume. The 10-mL injection volumes would produce a certain matrix effects, it could reduce matrix effects with the reduction of injection volume, the result of matrix effects could be satisfied when 2 mL volumes was injected into LC-MS.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
746
C. Wen et al.
When plasma sample processing method used diethyl ether, chloroform, methylene chloride or ethyl acetate for liquid–liquid extraction, it needs drying and reconstitution, the process is relatively cumbersome. In this study, treatment of plasma by protein precipitation is simple and it does not require reconstitution and drying process, although it will dilute the concentration of acetylcorynoline in plasma, but with the use of liquid chromatography mass spectrometry could meet the requirements of pharmacokinetic study. The solvent for precipitation method, methanol, acetonitrile and methanol–acetonitrile (10/90, v/v), the result found that acetonitrile was more effective than methanol. When acetonitrile added with 10% methanol, plasma could be completely wash off from the centrifuge tube, and it provided higher recovery, so that the present study used a methanol–acetonitrile (10/90, v/v) as a protein precipitant. Select carbamazepine as IS, because its chromatographic performance was similar with acetylcorynoline, and they had similar retention time; they were all weak alkaline substances, and the recoveries of them were similar, both suitable for detection in positive ion electrospray ionization interface.
Xenobiotica, 2014; 44(8): 743–748
Selectivity and matrix effect Figure 3 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with acetylcorynoline and IS, and a plasma sample. No interfering endogenous substance was observed at the retention time of the analyte and IS. The matrix effect for acetylcorynoline at concentrations of 10, 400 and 800 ng/mL were measured to be 90.3 ± 9.7%, 93.5 ± 5.1% and 88.7 ± 3.1% (n ¼ 6), respectively. The matrix effect for IS (200 ng/mL) was measured to be 102.1 ± 3.7% (n ¼ 6). As a result, matrix effect from plasma was negligible in this method. Calibration curve and sensitivity The linear regressions of the peak area ratios versus concentrations were fitted over the concentration range 5–1000 ng/mL for acetylcorynoline in rat plasma. Typical equation of the calibration curve was: y ¼ (0.00238 ± 0.00023) x + (0.02592 ± 0.02226), r ¼ 0.9971 ± 0.0016, where y represents the ratios of acetylcorynoline peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of acetylcorynoline in plasma was 5 ng/mL.
Figure 3. Representative LC-MS chromatograms of acetylcorynoline (1) and carbamazepine (IS, 2), (a) blank plasma; (b) blank plasma spiked with acetylcorynoline (LLOQ, 5 ng/mL) and IS (200 ng/mL), (c) a rat plasma sample 3 h after intravenous administration of single dosage 3 mg/kg acetylcorynoline (55 ng/mL).
LC-MS determination of acetylcorynoline in rat plasma
DOI: 10.3109/00498254.2014.887802
747
Table 1. Precision and accuracy for acetylcorynoline of QC sample in rat plasma (n¼6). Concentration (ng/mL) 5 10 400 800
CV (%)
Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
11.0 7.4 8.0 9.1
12.4 5.0 5.2 6.3
108.3 95.8 96.7 102.3
112.1 108.6 102.5 96.7
The precision and accuracy at LLOQ were 11.0% and 108.3%, respectively. The LOD, defined as a signal/noise ratio of 3, was 1.5 ng/mL for acetylcorynoline in rat plasma.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
Precision, accuracy and recovery The precision of the method was determined by calculating CV for QCs at three concentration levels over three validation days. Intra-day precision was 12% or less and the inter-day precision was 13% or less at each QC level. The accuracy of the method ranged from 95.8% to 112.1% at each QC level. Assay performance data are presented in Table 1. The results demonstrate that the values are within the acceptable range and the method is accurate and precise. Mean recoveries of acetylcorynoline at concentrations of 10, 400 and 800 ng/mL were measured to be 76.1 ± 7.4, 72.3 ± 6.4 and 87.6 ± 5.9% (n ¼ 6), respectively. The differences in the observed recovery rates at low–medium (72.3–76.1%) and high concentrations (87.6%), it may be an impact of the matrix, however the results of recovery could be acceptable. The recovery of the IS (200 ng/mL) was measured to be 93.4 ± 4.6%.
Figure 4. Mean plasma concentration time profile after intravenous administration of single dosage 3 mg/kg acetylcorynoline in six rats.
Carry-over
decreased very quickly, and the t1/2 was 1.8 h. The pharmacokinetic profile of acetylcorynoline in rat was characterized for the first time. It helps to better understand pharmacology features of acetylcorynoline.
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 four extra minutes to the end of the gradient elution effectively washed the system between samples thereby eliminating carry-over. Stability The auto-sampler, room temperature, freeze–thaw and longterm (20 days) stability results indicated that the analyte was stable under the storage conditions described above since the bias in concentration was within ±15% of their nominal values, and the established method was suitable for the pharmacokinetic study. Application to pharmacokinetics The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after intravenous administration of a single 3 mg/kg dose of acetylcorynoline was shown in Figure 4. The area under the plasma concentration–time curve (AUC), the mean residence time (MRT), the plasma clearance (CL), apparent volume of distribution (V), the maximum plasma concentration (Cmax) and the halflife (t1/2) were estimated using non-compartmental calculations performed with DAS (Drug and statistics) software. The main pharmacokinetic parameters were summarized in Table 2. In vivo, the acetylcorynoline concentration was
Table 2. The main pharmacokinetic parameters after intravenous administration of single dosage 3 mg/kg acetylcorynoline in six rats. Parameters
Unit
Mean (±SD)
AUC(0–t) AUC(0–1) MRT(0–t) MRT(0–1) t1/2 CL V Cmax
ng/mL h ng/mL h H H H L/h/kg L/kg ng/mL
426.2 ± 94.2 441.8 ± 98.4 1.7 ± 0.2 2.0 ± 0.2 1.8 ± 0.1 7.3 ± 1.5 19.4 ± 4.2 550.6 ± 21.6
Conclusion The LC-MS method for determination of acetylcorynoline in rat plasma utilizing 100 mL of plasma with an LLOQ of 5.0 ng/mL developed and validated, it was sensitive, selective and simple. A time-saving protein precipitation procedure made the method readily applicable in a further clinical study. The LC-MS method successfully applied to a pharmacokinetic study of acetylcorynoline after intravenous administration of single dosage 3 mg/kg to rats. The pharmacokinetic profile of acetylcorynoline in rat was characterized for the first time.
Declaration of interest The authors declare no conflict of interest.
References European Medicines Agency (EMA). (2011). Guideline on bioanalytical method validation. Available from: http://www.ema. europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/ 08/WC500109686.pdf [last accessed 7 Feb 2014]. Fu RH, Wang YC, Chen CS, et al. (2013a). Acetylcorynoline attenuates dopaminergic neuron degeneration and alpha-synuclein aggregation in
748
C. Wen et al.
Xenobiotica Downloaded from informahealthcare.com by University of Otago on 08/31/14 For personal use only.
animal models of Parkinson’s disease. Neuropharmacology. [Epub ahead of print]. doi:10.1016/j.neuropharm.2013.08.007. Fu RH, Wang YC, Liu SP, et al. (2013b). Acetylcorynoline impairs the maturation of mouse bone marrow-derived dendritic cells via suppression of IkappaB kinase and mitogen-activated protein kinase activities. PloS One 8:e58398. Ma J, Dong J, Lin G, et al. (2012). Determination of curdione in rabbit plasma by liquid chromatography mass spectrometry. Biomed Chromatogr 26:655–9. US Food and Drug Administration. (2001). Guidance for industry: bioanalytical method validation. Available from: http://www.fda.gov/ downloads/Drugs/Guidances/ucm070107.pdf [last accessed 7 Feb 2014]. Wang X, Dong H, Shu X, et al. (2012). Large-scale separation of alkaloids from Corydalis bungeana Turcz. by pH-zone-refining counter-current chromatography. Molecules 17:14968–74. Wang X, Xiang Z, Cai X, et al. (2011). Determination of pethidine in human plasma by LC-MS/MS. Biomed Chromatogr 25:833–7.
Xenobiotica, 2014; 44(8): 743–748
Wei HL, Liu GT. (1997). Protective action of corynoline, acetylcorynoline and protopine against experimental liver injury in mice. Acta Pharma Sin 32:331–6. Williams JS, Donahue SH, Gao H, Brummel CL. (2012). Universal LCMS method for minimized carryover in a discovery bioanalytical setting. Bioanalysis 4:1025–37. Xie C, Veitch NC, Houghton PJ, Simmonds MS. (2004). Flavonoid glycosides and isoquinolinone alkaloids from Corydalis bungeana. Phytochemistry 65:3041–7. Zhou Y, Li J, He X, et al. (2013a). Development and validation of a liquid chromatography-tandem mass spectrometry method for simultaneous determination of amlodipine, atorvastatin and its metabolites ortho-hydroxy atorvastatin and para-hydroxy atorvastatin in human plasma and its application in a bioequivalence study. J Pharm Biomed Anal 83C:101–7. Zhou Z, Wu X, Wei Q, et al. (2013b). Development and validation of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the simultaneous determination of five first-line antituberculosis drugs in plasma. Anal Bioanal Chem 405:6323–35.
