Journal of Chromatography B, 969 (2014) 12–18
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography–tandem mass spectrometry Bing Li a,b,c , Xu-Zheng Zhou a,b,c , Jian-Yong Li a,b,c , Ya-Jun Yang a,b,c , Jian-Rong Niu a,b,c , Xiao-Juan Wei a,b,c , Xi-Wang Liu a,b,c , Jin-Shan Li a,b,c , Ji-Yu Zhang a,b,c,∗ a
Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou, China Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou, China c Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS, Lanzhou 730050, China b
a r t i c l e
i n f o
Article history: Received 25 November 2013 Accepted 7 July 2014 Available online 8 August 2014 Keywords: Arecoline hydrobromide Arecoline hydrobromide tablets LC–MS/MS Pharmacokinetics Dog plasma
a b s t r a c t A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with -pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6 × 75 mm, 3.5 m) using methanol: 5 mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2–500 ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC–MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration. © 2014 Published by Elsevier B.V.
1. Introduction Taeniasis, a zoonotic parasitic disease, will cause serious health risk to humans and animals. The taeniasis/cysticercosis disease complex has been recognized as a re-emerging disease in both developed and developing countries [1,2]. Since 1990, several joint collaborative projects in China have been initiated to address not only the cestode zoonoses but also many other parasitic and/or infectious diseases [3]. Human cases of taeniasis and cysticercosis have been reported in Asia and the Asia–Pacific regions [4,5]. According to statistics, currently, dogs infected with Taeniasiscausing tapeworms in China and the Testour Region in Tunisiamore account for more than 30% and 27%, respectively [6]. Despite the large numbers of studies on this disease, efficient drugs to treat taeniasis remain to be developed. Praziquantel [7] was the most commonly used drug to treat taeniasis, however, its popularity has been lessened due to its pungent odor, which makes it difficult
∗ Corresponding author at: Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China. Tel.: +86 13893612415; fax: +86 931 2115191. E-mail address: [email protected] (J.-Y. Zhang). http://dx.doi.org/10.1016/j.jchromb.2014.07.043 1570-0232/© 2014 Published by Elsevier B.V.
for infected animals to swallow. Additionally, niclosamide, as an alternative drug, is severely toxic to some aquatic organisms [8,9]. Arecoline is a natural product, extracted from the plant species of Areca catechu L. Arecoline has antiparasitic, antifungaland antiviral effects, and is able to dissolve food stagnation and promote urination [10]. It has been widely used for treatment of tapeworm infestation, abdominal distension, diarrhea and edema [10]. Arecoline has high efficiency, low cost, and low toxicity, and is used in fruit form. However, the content of arecoline in extract from A. catechu L. is only 0.3% to 0.7% [11], which coupled with the limited sources of medicinal herbs and low efficiency of traditional extraction methods greatly limits its clinical application. On the other hand, arecoline as liquid is not convient to adminster, its’ salt is appropriate for clinic use. No formulations of arecoline in salt forms are currently available for clinical use. In our previous study, we have successfully synthesized bulk drug—arecoline hydrobromide and prepared arecoline hydrobromide tablets as an oral preparation for the first time. Acute toxicity studies indicated that the toxicity of arecoline is four times higher than that of arecoline hydrobromide, therefore, we synthesized the hydrobromide formulation [12]. The proposed synthesis process can achieve a yield of more than 78% [13]. The benefits of this new technology include not only high yield, but also easy purification of the product, simple process operation, and easy scale-up for industrial
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
13
a Milli-Q Plus water system (Millipore Corporation, Bedford, MA, USA) before use. 2.2. Equipment The LC–MS/MS equipment (1200-6410A) consisted of a LC system with a binary pump-SL and a triple quadrupole mass spectrometer with electrospray ionization (ESI) (Agilent Technologies, Inc., Santa Clara, CA, USA). Data were recorded, and the system was controlled using MassHunter software (version B.01.04, Agilent Technologies). 2.3. Chromatography and mass spectrometry conditions
Fig. 1. Chemical structures of arecoline and arecoline hydrobromide.
production [13]. Both arecoline hydrobromide drug and tablet are nontoxic and safe for clinical use according to acute toxicity studies of oral bulk arecoline hydrobromide drug and tablets in rats and mice [14,15]. In addition, arecoline hydrobromide can enhance bowel movements and increase digestive gland secretion for more rapid elimination of parasites [16], and has significant anthelmintic effects on tapeworms in dogs [17]. In order to define the pharmacokinetic profile of arecoline hydrobromide tablets, a new method for the characterization of arecoline is needed. As a result, we propose to use high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) to characterize the pharmacokinetics of arecoline in dog plasma. Previous attempts to characterize arecoline have mainly included acid–base titration, spectrophotometry, ion exchange chromatography [18], capillary electrophoresis (CE) [19], and high performance liquid chromatography [20]. Because arecoline contains no conjugated groups in the structure (Fig. 1), it has no appropriate chromophores for use in characterization techniques. Its maximum wavelength for ultraviolet absorption is 215 nm, which is near the end of the absorption range, and the sensitivity is thus low [21]. Therefore, we chose to develop a LC–MS/MS method for determining the concentration of arecoline in dog plasma. LC–MS/MS is accurate and sensitive, and requires only a simple protein precipitation pretreatment for arecoline with -pinene as an internal standard [22]. To our knowledge, this is the first report of an easy and reliable method for determining arecoline concentrations in dog plasma utilizing LC–MS/MS. Furthermore, we have successfully applied this method to characterize the pharmacokinetics of arecoline hydrobromide tablets in dogs following oral administration at 3 mg/kg. 2. Experimental
The separation was carried out at 25 ◦ C on a C18 column (4.6 × 75 mm, 3.5 m; Agilent Technologies). The mobile phase consisted of methanol:water (the water contained 5 mM ammonium acetate)(10:90, v/v) with a flow rate of 0.40 mL/min. The auto-sampler was conditioned at 4 ◦ C, and the injection volume was 1 L. The mass spectrometer was operated in positive ion mode with an ESI interface. Quantitation was performed by multiple reaction monitoring (MRM). In the positive mode, the MS/MS setting parameters were as follows: capillary voltage 4 kV, cone voltage 40 V, source temperature 100 ◦ C, and desolvation temperature 350 ◦ C with a desolvation nitrogen gas flow of 11 L/min and a cone gas flow of 8 L/min. The optimized fragmentation voltages for arecoline and IS were 92 V and 100 V, respectively, and the Delta electron multiplier voltage (EMV) was 200 V. Data were collected in multiple reaction monitoring (MRM) mode using transitions of m/z 156 → 53 (arecoline), with a collision energy of 25 eV, and m/z 137 → 107 (IS), with a collision energy of 20 eV. 2.4. Preparation of standard solutions and quality control samples A standard stock solution of arecoline hydrobromide was prepared by dissolving an accurately weighed, appropriate amount in pure water in a 10-mL volumetric flask to achieve a concentration of 100 g/mL. A standard stock solution of IS was prepared in anhydrous ethanol and diluted to 0.87 g/mL. A series of arecoline hydrobromide working standard solutions were prepared by dilutions of the stock solution with pure water to obtain the following concentrations: 2, 5, 10, 50, 200 and 500 ng/mL. All of the standard solutions were stored at 4 ◦ C and brought to room temperature before use. Plasma calibration standards of 2–500 ng/mL were prepared by spiking 10 L of each standard solution with an aliquot of 100 L blank dog plasma. Quality control (QC) samples were prepared in the same way at four concentrations of 2 ng/mL (QClower limit of quantitation [LLOQ]), 5 ng/mL (QC-low), 50 ng/mL (QC-med), and 500 ng/mL (QC-high). Both the calibration standard and the QC samples were used in the method validation and the pharmacokinetic study.
2.1. Chemicals 2.5. Sample preparation Arecoline hydrobromide was provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) with batch number of 111684-200401 and purity >99%. Internal standard (IS) -pinene was purchased from TCI Co., Ltd. (Tokyo, Japan). Arecoline hydrobromide tablets were supplied by Lanzhou Institute of Animal Science and Veterinary Pharmaceutics. Methanol (HPLC grade) was purchased from Fisher Chemical (Waltham, USA). All other chemicals were of analytical grade and were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Ningbo, China). Pure water was obtained by purification through
Plasma aliquots (100 L) were spiked with 10 L of methanol and -pinene (10 L of 0.87 g/mL solution) as an internal standard and mixed (when preparing calibration and QC samples, standard solution was added instead of methanol). Then, 20 L of AgNO3 solution (0.1 M) and 0.4 mg of NaCl were added followed by vortex mixing for 2 min, and 10 L of NaOH (1 M) was added followed by vortex mixing for 1 min. Then, 300 L of N-hexane (containing 1% isoamyl alcohol) was added followed by vortex mixing for another 2 min for protein precipitation. Finally, samples were centrifuged at
14
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
Table 1 Intra- and inter-day precision and accuracy of arecoline (n = 6) in dog plasma. Concentration (ng/mL)
Intra-day precision and accuracy (n = 6) Accuracy (%) ± SD
5 50 500 2
93.7 94.3 97.5 91.6
± ± ± ±
3.1 2.3 1.8 2.4
4000 rpm for 20 min. The supernatant was transferred and evaporated to dryness under a stream of N2 at 40 ◦ C. The dried extract was resuspended in 100 L of pure water for injection into the LC–MS/MS system. Each sample was transferred to an injection vial after filtering through a 0.45-m Millipore filter, and 1 L of this solution was injected into the LC–MS/MS system for quantitative analysis. 2.6. Validation The LC–MS/MS method was validated with respect to linearity, selectivity, LLOQ, recovery, matrix effect, intra- and interday accuracy and precision, and stability of the analyte during the sample storage and processing procedures. 2.6.1. Selectivity Selectivity was evaluated by comparing the chromatograms of six different blank plasma samples obtained from six subjects with reference to those spiked with arecoline hydrobromide and IS. 2.6.2. Linearity and LLOQ A calibration curve was constructed from plasma standards at six concentrations of arecoline ranging from 2 to 500 ng/mL. The curve was constructed by plotting the peak area ratio of arecoline/IS versus the nominal concentration of arecoline. The correlation coefficient and linear regression equation were used to calculate the analyte concentration in the samples. A weighted (1/×2) linear least-squares regression was used as the mathematical model. The lowest concentration that produced an S/N of 5 was considered the LLOQ [23]. The concentration that produced an S/N of 3 was considered the limit of detection (LOD) [23]. 2.6.3. Precision and accuracy Intra-day accuracy and precision were evaluated by analyzing the QC levels at four concentrations (2, 5, 50, and 500 ng/mL; Table 1) with six determinations per concentration on the same day. The interday accuracy and precision were evaluated by the analysis of the QC levels at the same four concentrations with six determinations per concentration over 3 days. Precision and accuracy were based on the criteria that the relative standard deviation (RSD) for each concentration should not exceed 15%, except for the LLOQ, which should not exceed 20% [24].
Inter-day precision and accuracy (n = 6) RSD (%)
Accuracy (%) ± SD
3.3 2.4 1.8 2.6
92.5 94.9 96.8 90.3
± ± ± ±
2.2 2.0 1.5 2.8
RSD (%) 2.4 2.1 2.1 3.1
stability of arecoline in dog plasma was assessed by analyzing replicates (n = 6) of QC samples at concentrations of 5, 50, and 500 ng/mL during the sample storage and processing procedures. The stability of stock solutions of arecoline was analyzed after 48 h at room temperature and after 1 month at 4 ◦ C. Short-term stability was assessed after exposure of the plasma samples to ambient temperature for 24 h. Long-term stability was assessed after storage of the plasma samples at −20 ◦ C for 30 days. The freeze/thaw stability was determined after three freeze/thaw cycles (room temperature to −20 ◦ C). Sample stability in the autosampler tray was evaluated after 48 h at 4 ◦ C, which simulates the residence time of the samples in the autosampler for each analytical run. 2.7. Pharmacokinetic study The assay method described above was applied to study the pharmacokinetics of arecoline hydrobromide in dog plasma after oral administration in tablet form. All the experimental procedures were approved by and performed in accordance with the guidelines of the Care and Use of Laboratory Animals of the Lanzhou Institute of Animal Science and Veterinary Pharmaceutics. Healthy beagles (9.7 ± 1.1 kg) were obtained from DiLePu Biological Resources Development Co., Ltd. of Xi’an (Xi’an, Shanxi, China) and housed in standard environmentally controlled animal rooms (temperature: 25 ± 2 ◦ C, humidity: 50 ± 20%) with a natural light–dark cycle for 1 week before experiments. The dogs were fasted for 12 h before dosing but allowed free movement and access to water throughout the experiments. The dogs were fed a diluted tincture of iodine (5 mL) to prevent vomiting before dosing. All dogs (n = 12) were dosed orally at a dosage of 3 mg/kg. After a single dose was administered by oral administration, blood samples (4 mL) were collected in heparinized tubes via the jugular vein after 0.5, 0.67, 0.83, 1, 1.25, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 h. After all blood samples were centrifuged at 12,000 rpm for 10 min, the plasma samples were collected and then immediately stored in a −20 ◦ C freezer until analysis using LC–MS/MS. The pharmacokinetic parameters were calculated using WinNonlin professional software version 5.2 (Pharsight, Mountain View, CA, USA) and the one-compartment model. The half-life (t1/2 ) and absorption half-life (t1/2a ) were calculated directly according to the pharmacokinetic parameters. 3. Results and discussion
2.6.4. Recovery and matrix effect The recovery was determined in quadruplicate by comparing processed QC samples at three levels (low, medium, and high) with reference solutions in blank plasma samples at the same levels. The matrix effect was determined by comparing the peak areas of the post-extracted spiked samples with those of the standards containing equivalent amounts of arecoline prepared in the mobile phase. The experiments were performed with six batches per each of the three levels. 2.6.5. Stability For all stability experiments, freshly prepared QC samples were analyzed with reference to a freshly prepared standard curve. The
3.1. Mass spectrometric detection To optimize the positive ESI mode conditions, arecoline hydrobromide was dissolved in pure water and IS was dissolved in anhydrous ethanol. The solutions were then injected into the mass spectrometer for scanning in the positive ion mode. When arecoline and IS were injected directly into the mass spectrometer along with the mobile phase, the analytes yielded predominantly [M + H]+ ions at m/z 156.2 for arecoline (Fig. 2) and at m/z 137.2 for IS. Each of the precursor ions was subjected to collision-induced dissociation to determine the resulting product ions from the product ion mass spectra, and the most abundant and stable fragment ions were
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
15
Fig. 2. Full mass spectra scan for arecoline (A), product ions for arecoline (B), and MRM for arecoline (C).
generated at m/z 53.0 for arecoline and m/z 107.0 for IS. Therefore, the mass transitions chosen for quantitation were m/z 156.2 to 53.0 for arecoline and m/z 137.2 to 107.0 for IS [22]. 3.2. Chromatographic separation A 75-mm column subjected to an isocratic flow rate of 0.4 mL/min for less than 3 min was used for chromatographic separation in this study. The mobile phase, which consisted of a mixture of methanol: 5 mM ammonium acetate (10:90, v/v), was found to be suitable for separation and ionization of arecoline and IS. In addition, the presence of ammonium acetate was found to improved the ionization of the compounds. Under optimized LC and MS conditions, arecoline and IS compounds were separated with retention times of 0.59 and 2.37 min, respectively (Fig. 3).
the solution to adjust the pH value. Protein precipitation was chosen for sample pretreatment, and N-hexane containing 1% isoamyl alcohol was chosen as a precipitant in this study, as it ensures good clean-up of the plasma samples, high recovery, high sensitivity, and effective elimination of the matrix effect. 3.4. Method validation 3.4.1. Selectivity The selectivity of the method was evaluated by analyzing individual blank plasma samples from six different sources. All samples were found to have no interference from endogenous substances affecting the retention time of either arecoline or IS. There was good base-line separation of the arecoline and IS extracted from the dog plasma. Representative chromatograms of blank plasma and blank plasma spiked with both arecoline and IS are shown in Fig. 3.
3.3. Sample preparation Since arecoline has a higher recovery than arecoline hydrobromide, AgNO3 was added to react with arecoline hydrobromide, which would produce free arecoline for final quantitative analysis. The precipitate AgBr was then removed, and NaOH was added to
3.4.2. Linearity and LLOQ A calibration curve was constructed from plasma standards at six arecoline concentrations, ranging from 2 to 500 ng/mL. The ratio of peak areas of arecoline to that of IS was used for quantification. The calibration model was selected based on analysis of the
16
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
Fig. 3. Chromatograms for (A) blank plasma and (B) arecoline (a) and IS (b) in dog plasma.
data by linear regression with intercepts and a 1/×2 weighting factor. A typical equation for the arecoline calibration curve was: y = 0.0043x + 0.0361, (r2 = 0.9993), where y is the peak-area ratio of arecoline to IS and x is the plasma concentration of arecoline. The calibration curve is shown in Fig. 4. The LLOQ for arecoline was established to be 2 ng/mL, with an accuracy of 98.8%. The LOD was found to be 0.2 ng/mL arecoline. 3.4.3. Accuracy and precision The intra- and inter-day precision and accuracy for the QC samples (2, 5, 50, and 500 ng/mL) are summarized in Table 1. These data demonstrated that the developed method has satisfactory accuracy, precision, and reproducibility for the quantification of arecoline concentration in plasma.
3.4.4. Recovery and matrix effect The mean extraction recoveries of arecoline were 91.0 ± 2.8%, 94.8 ± 2.4%, and 95.5 ± 2.3% at concentrations of 5, 50, and 500 ng/mL, respectively. The mean extraction recovery of IS was 95.6 ± 2.4% at 100 ng/mL. These results suggest that the recovery of arecoline was consistent and not concentration-dependent. Recovery values are listed in Table 2. The matrix effects ranged from 92.8 ± 2.8% to 97.4 ± 2.0% for arecoline at concentrations of 5, 50, and 500 ng/mL, whereas the matrix effect for IS (100 ng/mL) was 92.5 ± 2.2%. Therefore, limited matrix effects were observed. 3.4.5. Stability The stability of arecoline was tested under freeze/thaw, shortterm, autosampler, and long-term conditions (Table 3). No stability issues were observed in any of the experiments. Arecoline was stable in dog plasma for three freeze/thaw cycles, at room temperature for 48 h, in the autosampler tray at 4 ◦ C for 48 h, and when Table 2 Recovery of arecoline (n = 6) from dog plasma. Spiked concentration (ng/mL)
5 50 500
Fig. 4. Calibration curve.
Recovery (%, n = 6)a Mean ± SD
RSD (%)b
91.0 ± 2.8 94.8 ± 2.4 95.5 ± 2.3
3.1 2.5 2.4
a Recovery = ratio of response of spiked standard before extraction to that after extraction. b RSD, relative standard deviation.
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18 Table 3 Stability of arecoline in dog plasma samples under various conditions (n = 6). Storage conditions At room temperature for 48 h At 4 ◦ C for 1 month
At -20 ◦ C for 30 days
At 4 ◦ C in the autosampler for 48 h 3 freeze-thaw cycles
Concentration (%) (ng/mL)
Accuracy (%) ±SD
5 50 500 5 50 500 5 50 500 5 50 500 5 50 500
103.5 103.1 97.2 93.6 96.7 96.4 94.1 95.5 95.9 92.7 97.6 96.3 91.3 95.6 95.8
Table 4 Pharmacokinetic parameters of arecoline after oral administration (n = 12). RSD
Parameter a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.9 2.3 2.1 2.6 1.8 2.1 2.0 2.2 1.5 2.2 2.3 1.9 2.8 3.0 2.1
17
1.8 2.2 2.2 2.8 1.9 2.2 2.1 2.3 1.6 2.4 2.4 2.0 3.1 3.1 2.2
AUC0–t (min ng/mL) AUC0–∞ (min ng/mL) b K (1/min) c Ka (1/min) d Cmax (ng/mL) e Tmax (min) f CL/F (L/min/kg) g t1/2 (min) h t1/2a (min) a b c d e f g h
Mean 15,116.86 15,771.37 0.01 0.014 60.61 120.07 0.19 69.32 49.93
CV% 19.96 18.24 13.36 11.98 0.01 0.01 15.38 9.7 7.69
AUC, area under the concentration–time curve. K, elimination rate constant. Ka , absorption rate constant. Cmax , peak plasma concentration. Tmax , peak time. CL/F , plasma clearance. t1/2 , half life. t1/2a , absorption half life.
−20 ◦ C
stored at for 30 days. Arecoline was also stable in stock solutions at room temperature for 48 h and at 4 ◦ C for 1 month. Based on these results, we concluded that arecoline can be stored and processed under routine laboratory conditions without special considerations. 3.5. Application to pharmacokinetic study The present method was successfully validated and then applied to quantitate arecoline in plasma samples after oral administration of arecoline hydrobromide tablets to beagle at a dose of 3 mg/kg. No previous studies of arecoline hydrobromide concentration in plasma samples or related pharmacokinetic studies had been reported. In our study, all samples were available and could be analyzed. The sensitivity and specificity of the assay were found to be sufficient for accurately characterizing the plasma pharmacokinetics of arecoline in dogs. After oral administration of the preparation, close and continuous visual monitoring of the animals revealed that there were no side effects. Fig. 5 shows the mean plasma concentration vs. time profile for arecoline hydrobromide in dogs after oral administration of arecoline hydrobromide tablets. The major pharmacokinetic parameters were calculated by use of a one-compartment model with first order absorption and are listed in Table 4. Tmax and Cmax were 120.07 min and 60.61 ng/mL, respectively for arecoline, and the t1/2 of arecoline was 69.32 min. The area under the concentration–time curve values, AUC0–t and AUC0–∞ , were 15,116.86 min ng/mL and 15,771.37 min ng/mL, respectively, and the CL/F of arecoline was 0.19 L/min/kg. These results indicated that arecoline was rapidly absorbed and eliminated from the blood of the dogs (Fig. 5). No significant differences were found between
male and female dogs (data not shown). This study provided useful information to serve as a basis for further research on treatments using arecoline hydrobromide tablets. Method development and evaluation of the pharmacokinetic properties of this formulation will aid the preparation of new formulations of similar drugs with improved pharmacokinetic profiles. 4. Conclusions Pharmacokinetic analysis of arecoline hydrobromide tablets requires a highly sensitive assay that can determine the arecoline concentration in plasma after oral administration. Limited volumes of plasma available and interference from the biological matrix add to the complexity of the trace analysis of arecoline. In this study, a rapid and sensitive LC–MS/MS method was developed and validated, and then successfully applied to evaluate the pharmacokinetic parameters of arecoline after oral administration of arecoline hydrobromide tablets to dog. Sample preparation is simple and relatively quick, and the analysis requires only 100 L of plasma and a short run time of less than 3 min, which is very advantageous in a pharmacokinetic study. The method has excellent sensitivity, linearity, precision, and accuracy. Currently, no bulk drug or other formulations of arecoline hydrobromide are available for clinical use. The proposed LC–MS/MS assay is an excellent technique for further evaluating the pharmacokinetic properties and therapeutic potential of the new preparation of arecoline hydrobromide tablets as a taenifuge. This study provided helpful references for the future clinical application of this herb. Acknowledgments This work was economically supported by the earmarked fund of the China Agriculture Research System (cars-38) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201303038-4). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.07.043. References
Fig. 5. Mean plasma concentration–time profile after oral administration of arecoline hydrobromide tablets to dogs (n = 12) at 3 mg/kg.
[1] G.M. Simanjuntak, S.S. Margono, M. Okamoto, A. Ito, Parasitol. Today 13 (1997) 321.
18
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
[2] P.M. Schantz, P.P. Wilkins, V.C.W. Tsange, Emerging Infections 2, ASM Press, Washington, DC, 1998. [3] A. Ito, Parasitol. Int. 55 (Suppl) (2006) S3. [4] A. Ito, M. Nakao, T. Wandra, Lancet 362 (2003) 1918. [5] A. Ito, P.S. Craig, P.M. Schantz, Parasitol. Int. 55 (Suppl) (2006) S1. [6] S. Lahmar, M. Kilani, P.R. Torgerson, Ann. Trop. Med. Parasitol. 95 (2001) 69. [7] S.H. Xiao, Acta Trop. 96 (2005) 153. [8] B.M. Al-Hadiya, Profiles Drug Subst. Excip. Relat. Methodol. 32 (2005) 67. [9] F.C. Abreu, M.O. Goulart, A.M. Brett, Biosens. Bioelectron. 17 (2002) 913. [10] Z. Cai, Y. Li, L. Li, Z. Chen, J. Pharm. Anal. 2 (2012) 356. [11] J.C. He, Z.C. Dong, J.R. Wang, Chin. Arch. Tradit. Chin. Med. 29 (2011) 1967. [12] C.Y. Zhao, J.Y. Zhang, X.Z. Zhou, Y.M. Wei, J.Y. Li, Z.F. Zhang, J.S. Li, X.J. Wei, J.R. Niu, J. Tradit. Chin. Vet. Med. 26 (2007) 33. [13] Z.F. Zhang, Chinese Academy of Agricultural Sciences, Beijing, 2007.
[14] X.Z. Zhou, J.Y. Zhang, C.Y. Zhao, J.S. Li, Z.F. Zhang, J.Y. Li, X.J. Wei, J.R. Niu, L. Wang, Progr. Vet. Med. 28 (2007) 34. [15] X.Z. Zhou, J.Y. Zhang, J.S. Li, Heilongjiang Anim. Sci. Vet. Med. 21 (2012) 114. [16] X.Z. Zhou, J.Y. Zhang, C.Y. Zhao, Chin. J. Vet. Med. 41 (2007) 28. [17] X.Z. Zhou, J.Y. Zhang, J.Y. Li, J. Anhui Agric. Sci. 38 (2010) 13119, 13517. [18] H.A. Chen, Y.H. Lai, X.Y. Wang, W.R. Yang, J. Chin. Med. Mater. 25 (2002) 27. [19] W. Yuan, J.D. Lu, X.Y. Fu, Chin. J. Anal. Chem. 28 (2000) 749. [20] S. Cox, I. Piatkov, E.R. Vickers, G. Ma, J. Chromatogr. A 1032 (2004) 93. [21] J. Huang, M. McLeish, J. Chromatogr. 475 (1989) 447. [22] F.L. Ding, S.L. Peng, Acad. Period. Farm Prod. Process. 4 (2008) 77. [23] D. Pabbisetty, A. Illendula, K.M. Muraleedharan, A.G. Chittiboyina, J.S. Williamsonb, M.A. Avery, B.A. Avery, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 889–890 (2012) 125. [24] H. Fan, R. Li, Y. Gu, D. Si, C. Liu, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 889–890 (2012) 105.
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination and pharmacokinetic studies of arecoline in dog plasma by liquid chromatography–tandem mass spectrometry Bing Li a,b,c , Xu-Zheng Zhou a,b,c , Jian-Yong Li a,b,c , Ya-Jun Yang a,b,c , Jian-Rong Niu a,b,c , Xiao-Juan Wei a,b,c , Xi-Wang Liu a,b,c , Jin-Shan Li a,b,c , Ji-Yu Zhang a,b,c,∗ a
Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture, Lanzhou, China Key Laboratory of New Animal Drug Project of Gansu Province, Lanzhou, China c Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS, Lanzhou 730050, China b
a r t i c l e
i n f o
Article history: Received 25 November 2013 Accepted 7 July 2014 Available online 8 August 2014 Keywords: Arecoline hydrobromide Arecoline hydrobromide tablets LC–MS/MS Pharmacokinetics Dog plasma
a b s t r a c t A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of arecoline concentration in dog plasma. Plasma sample was prepared by protein precipitation using n-hexane (containing 1% isoamyl alcohol) with -pinene as an internal standard. Chromatographic separation was achieved on an Agilent C18 column (4.6 × 75 mm, 3.5 m) using methanol: 5 mM ammonium acetate as the mobile phase with isocratic elution. Mass detection was carried out using positive electrospray ionization in multiple reaction monitoring mode. The calibration curve for arecoline was linear over a concentration range of 2–500 ng/mL. The intra-day and inter-day accuracy and precision were within the acceptable limits of ±10% at all concentrations. In summary, the LC–MS/MS method described herein was fully validated and successfully applied to the pharmacokinetic study of arecoline hydrobromide tablets in dogs after oral administration. © 2014 Published by Elsevier B.V.
1. Introduction Taeniasis, a zoonotic parasitic disease, will cause serious health risk to humans and animals. The taeniasis/cysticercosis disease complex has been recognized as a re-emerging disease in both developed and developing countries [1,2]. Since 1990, several joint collaborative projects in China have been initiated to address not only the cestode zoonoses but also many other parasitic and/or infectious diseases [3]. Human cases of taeniasis and cysticercosis have been reported in Asia and the Asia–Pacific regions [4,5]. According to statistics, currently, dogs infected with Taeniasiscausing tapeworms in China and the Testour Region in Tunisiamore account for more than 30% and 27%, respectively [6]. Despite the large numbers of studies on this disease, efficient drugs to treat taeniasis remain to be developed. Praziquantel [7] was the most commonly used drug to treat taeniasis, however, its popularity has been lessened due to its pungent odor, which makes it difficult
∗ Corresponding author at: Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China. Tel.: +86 13893612415; fax: +86 931 2115191. E-mail address: [email protected] (J.-Y. Zhang). http://dx.doi.org/10.1016/j.jchromb.2014.07.043 1570-0232/© 2014 Published by Elsevier B.V.
for infected animals to swallow. Additionally, niclosamide, as an alternative drug, is severely toxic to some aquatic organisms [8,9]. Arecoline is a natural product, extracted from the plant species of Areca catechu L. Arecoline has antiparasitic, antifungaland antiviral effects, and is able to dissolve food stagnation and promote urination [10]. It has been widely used for treatment of tapeworm infestation, abdominal distension, diarrhea and edema [10]. Arecoline has high efficiency, low cost, and low toxicity, and is used in fruit form. However, the content of arecoline in extract from A. catechu L. is only 0.3% to 0.7% [11], which coupled with the limited sources of medicinal herbs and low efficiency of traditional extraction methods greatly limits its clinical application. On the other hand, arecoline as liquid is not convient to adminster, its’ salt is appropriate for clinic use. No formulations of arecoline in salt forms are currently available for clinical use. In our previous study, we have successfully synthesized bulk drug—arecoline hydrobromide and prepared arecoline hydrobromide tablets as an oral preparation for the first time. Acute toxicity studies indicated that the toxicity of arecoline is four times higher than that of arecoline hydrobromide, therefore, we synthesized the hydrobromide formulation [12]. The proposed synthesis process can achieve a yield of more than 78% [13]. The benefits of this new technology include not only high yield, but also easy purification of the product, simple process operation, and easy scale-up for industrial
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
13
a Milli-Q Plus water system (Millipore Corporation, Bedford, MA, USA) before use. 2.2. Equipment The LC–MS/MS equipment (1200-6410A) consisted of a LC system with a binary pump-SL and a triple quadrupole mass spectrometer with electrospray ionization (ESI) (Agilent Technologies, Inc., Santa Clara, CA, USA). Data were recorded, and the system was controlled using MassHunter software (version B.01.04, Agilent Technologies). 2.3. Chromatography and mass spectrometry conditions
Fig. 1. Chemical structures of arecoline and arecoline hydrobromide.
production [13]. Both arecoline hydrobromide drug and tablet are nontoxic and safe for clinical use according to acute toxicity studies of oral bulk arecoline hydrobromide drug and tablets in rats and mice [14,15]. In addition, arecoline hydrobromide can enhance bowel movements and increase digestive gland secretion for more rapid elimination of parasites [16], and has significant anthelmintic effects on tapeworms in dogs [17]. In order to define the pharmacokinetic profile of arecoline hydrobromide tablets, a new method for the characterization of arecoline is needed. As a result, we propose to use high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) to characterize the pharmacokinetics of arecoline in dog plasma. Previous attempts to characterize arecoline have mainly included acid–base titration, spectrophotometry, ion exchange chromatography [18], capillary electrophoresis (CE) [19], and high performance liquid chromatography [20]. Because arecoline contains no conjugated groups in the structure (Fig. 1), it has no appropriate chromophores for use in characterization techniques. Its maximum wavelength for ultraviolet absorption is 215 nm, which is near the end of the absorption range, and the sensitivity is thus low [21]. Therefore, we chose to develop a LC–MS/MS method for determining the concentration of arecoline in dog plasma. LC–MS/MS is accurate and sensitive, and requires only a simple protein precipitation pretreatment for arecoline with -pinene as an internal standard [22]. To our knowledge, this is the first report of an easy and reliable method for determining arecoline concentrations in dog plasma utilizing LC–MS/MS. Furthermore, we have successfully applied this method to characterize the pharmacokinetics of arecoline hydrobromide tablets in dogs following oral administration at 3 mg/kg. 2. Experimental
The separation was carried out at 25 ◦ C on a C18 column (4.6 × 75 mm, 3.5 m; Agilent Technologies). The mobile phase consisted of methanol:water (the water contained 5 mM ammonium acetate)(10:90, v/v) with a flow rate of 0.40 mL/min. The auto-sampler was conditioned at 4 ◦ C, and the injection volume was 1 L. The mass spectrometer was operated in positive ion mode with an ESI interface. Quantitation was performed by multiple reaction monitoring (MRM). In the positive mode, the MS/MS setting parameters were as follows: capillary voltage 4 kV, cone voltage 40 V, source temperature 100 ◦ C, and desolvation temperature 350 ◦ C with a desolvation nitrogen gas flow of 11 L/min and a cone gas flow of 8 L/min. The optimized fragmentation voltages for arecoline and IS were 92 V and 100 V, respectively, and the Delta electron multiplier voltage (EMV) was 200 V. Data were collected in multiple reaction monitoring (MRM) mode using transitions of m/z 156 → 53 (arecoline), with a collision energy of 25 eV, and m/z 137 → 107 (IS), with a collision energy of 20 eV. 2.4. Preparation of standard solutions and quality control samples A standard stock solution of arecoline hydrobromide was prepared by dissolving an accurately weighed, appropriate amount in pure water in a 10-mL volumetric flask to achieve a concentration of 100 g/mL. A standard stock solution of IS was prepared in anhydrous ethanol and diluted to 0.87 g/mL. A series of arecoline hydrobromide working standard solutions were prepared by dilutions of the stock solution with pure water to obtain the following concentrations: 2, 5, 10, 50, 200 and 500 ng/mL. All of the standard solutions were stored at 4 ◦ C and brought to room temperature before use. Plasma calibration standards of 2–500 ng/mL were prepared by spiking 10 L of each standard solution with an aliquot of 100 L blank dog plasma. Quality control (QC) samples were prepared in the same way at four concentrations of 2 ng/mL (QClower limit of quantitation [LLOQ]), 5 ng/mL (QC-low), 50 ng/mL (QC-med), and 500 ng/mL (QC-high). Both the calibration standard and the QC samples were used in the method validation and the pharmacokinetic study.
2.1. Chemicals 2.5. Sample preparation Arecoline hydrobromide was provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) with batch number of 111684-200401 and purity >99%. Internal standard (IS) -pinene was purchased from TCI Co., Ltd. (Tokyo, Japan). Arecoline hydrobromide tablets were supplied by Lanzhou Institute of Animal Science and Veterinary Pharmaceutics. Methanol (HPLC grade) was purchased from Fisher Chemical (Waltham, USA). All other chemicals were of analytical grade and were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Ningbo, China). Pure water was obtained by purification through
Plasma aliquots (100 L) were spiked with 10 L of methanol and -pinene (10 L of 0.87 g/mL solution) as an internal standard and mixed (when preparing calibration and QC samples, standard solution was added instead of methanol). Then, 20 L of AgNO3 solution (0.1 M) and 0.4 mg of NaCl were added followed by vortex mixing for 2 min, and 10 L of NaOH (1 M) was added followed by vortex mixing for 1 min. Then, 300 L of N-hexane (containing 1% isoamyl alcohol) was added followed by vortex mixing for another 2 min for protein precipitation. Finally, samples were centrifuged at
14
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
Table 1 Intra- and inter-day precision and accuracy of arecoline (n = 6) in dog plasma. Concentration (ng/mL)
Intra-day precision and accuracy (n = 6) Accuracy (%) ± SD
5 50 500 2
93.7 94.3 97.5 91.6
± ± ± ±
3.1 2.3 1.8 2.4
4000 rpm for 20 min. The supernatant was transferred and evaporated to dryness under a stream of N2 at 40 ◦ C. The dried extract was resuspended in 100 L of pure water for injection into the LC–MS/MS system. Each sample was transferred to an injection vial after filtering through a 0.45-m Millipore filter, and 1 L of this solution was injected into the LC–MS/MS system for quantitative analysis. 2.6. Validation The LC–MS/MS method was validated with respect to linearity, selectivity, LLOQ, recovery, matrix effect, intra- and interday accuracy and precision, and stability of the analyte during the sample storage and processing procedures. 2.6.1. Selectivity Selectivity was evaluated by comparing the chromatograms of six different blank plasma samples obtained from six subjects with reference to those spiked with arecoline hydrobromide and IS. 2.6.2. Linearity and LLOQ A calibration curve was constructed from plasma standards at six concentrations of arecoline ranging from 2 to 500 ng/mL. The curve was constructed by plotting the peak area ratio of arecoline/IS versus the nominal concentration of arecoline. The correlation coefficient and linear regression equation were used to calculate the analyte concentration in the samples. A weighted (1/×2) linear least-squares regression was used as the mathematical model. The lowest concentration that produced an S/N of 5 was considered the LLOQ [23]. The concentration that produced an S/N of 3 was considered the limit of detection (LOD) [23]. 2.6.3. Precision and accuracy Intra-day accuracy and precision were evaluated by analyzing the QC levels at four concentrations (2, 5, 50, and 500 ng/mL; Table 1) with six determinations per concentration on the same day. The interday accuracy and precision were evaluated by the analysis of the QC levels at the same four concentrations with six determinations per concentration over 3 days. Precision and accuracy were based on the criteria that the relative standard deviation (RSD) for each concentration should not exceed 15%, except for the LLOQ, which should not exceed 20% [24].
Inter-day precision and accuracy (n = 6) RSD (%)
Accuracy (%) ± SD
3.3 2.4 1.8 2.6
92.5 94.9 96.8 90.3
± ± ± ±
2.2 2.0 1.5 2.8
RSD (%) 2.4 2.1 2.1 3.1
stability of arecoline in dog plasma was assessed by analyzing replicates (n = 6) of QC samples at concentrations of 5, 50, and 500 ng/mL during the sample storage and processing procedures. The stability of stock solutions of arecoline was analyzed after 48 h at room temperature and after 1 month at 4 ◦ C. Short-term stability was assessed after exposure of the plasma samples to ambient temperature for 24 h. Long-term stability was assessed after storage of the plasma samples at −20 ◦ C for 30 days. The freeze/thaw stability was determined after three freeze/thaw cycles (room temperature to −20 ◦ C). Sample stability in the autosampler tray was evaluated after 48 h at 4 ◦ C, which simulates the residence time of the samples in the autosampler for each analytical run. 2.7. Pharmacokinetic study The assay method described above was applied to study the pharmacokinetics of arecoline hydrobromide in dog plasma after oral administration in tablet form. All the experimental procedures were approved by and performed in accordance with the guidelines of the Care and Use of Laboratory Animals of the Lanzhou Institute of Animal Science and Veterinary Pharmaceutics. Healthy beagles (9.7 ± 1.1 kg) were obtained from DiLePu Biological Resources Development Co., Ltd. of Xi’an (Xi’an, Shanxi, China) and housed in standard environmentally controlled animal rooms (temperature: 25 ± 2 ◦ C, humidity: 50 ± 20%) with a natural light–dark cycle for 1 week before experiments. The dogs were fasted for 12 h before dosing but allowed free movement and access to water throughout the experiments. The dogs were fed a diluted tincture of iodine (5 mL) to prevent vomiting before dosing. All dogs (n = 12) were dosed orally at a dosage of 3 mg/kg. After a single dose was administered by oral administration, blood samples (4 mL) were collected in heparinized tubes via the jugular vein after 0.5, 0.67, 0.83, 1, 1.25, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 h. After all blood samples were centrifuged at 12,000 rpm for 10 min, the plasma samples were collected and then immediately stored in a −20 ◦ C freezer until analysis using LC–MS/MS. The pharmacokinetic parameters were calculated using WinNonlin professional software version 5.2 (Pharsight, Mountain View, CA, USA) and the one-compartment model. The half-life (t1/2 ) and absorption half-life (t1/2a ) were calculated directly according to the pharmacokinetic parameters. 3. Results and discussion
2.6.4. Recovery and matrix effect The recovery was determined in quadruplicate by comparing processed QC samples at three levels (low, medium, and high) with reference solutions in blank plasma samples at the same levels. The matrix effect was determined by comparing the peak areas of the post-extracted spiked samples with those of the standards containing equivalent amounts of arecoline prepared in the mobile phase. The experiments were performed with six batches per each of the three levels. 2.6.5. Stability For all stability experiments, freshly prepared QC samples were analyzed with reference to a freshly prepared standard curve. The
3.1. Mass spectrometric detection To optimize the positive ESI mode conditions, arecoline hydrobromide was dissolved in pure water and IS was dissolved in anhydrous ethanol. The solutions were then injected into the mass spectrometer for scanning in the positive ion mode. When arecoline and IS were injected directly into the mass spectrometer along with the mobile phase, the analytes yielded predominantly [M + H]+ ions at m/z 156.2 for arecoline (Fig. 2) and at m/z 137.2 for IS. Each of the precursor ions was subjected to collision-induced dissociation to determine the resulting product ions from the product ion mass spectra, and the most abundant and stable fragment ions were
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
15
Fig. 2. Full mass spectra scan for arecoline (A), product ions for arecoline (B), and MRM for arecoline (C).
generated at m/z 53.0 for arecoline and m/z 107.0 for IS. Therefore, the mass transitions chosen for quantitation were m/z 156.2 to 53.0 for arecoline and m/z 137.2 to 107.0 for IS [22]. 3.2. Chromatographic separation A 75-mm column subjected to an isocratic flow rate of 0.4 mL/min for less than 3 min was used for chromatographic separation in this study. The mobile phase, which consisted of a mixture of methanol: 5 mM ammonium acetate (10:90, v/v), was found to be suitable for separation and ionization of arecoline and IS. In addition, the presence of ammonium acetate was found to improved the ionization of the compounds. Under optimized LC and MS conditions, arecoline and IS compounds were separated with retention times of 0.59 and 2.37 min, respectively (Fig. 3).
the solution to adjust the pH value. Protein precipitation was chosen for sample pretreatment, and N-hexane containing 1% isoamyl alcohol was chosen as a precipitant in this study, as it ensures good clean-up of the plasma samples, high recovery, high sensitivity, and effective elimination of the matrix effect. 3.4. Method validation 3.4.1. Selectivity The selectivity of the method was evaluated by analyzing individual blank plasma samples from six different sources. All samples were found to have no interference from endogenous substances affecting the retention time of either arecoline or IS. There was good base-line separation of the arecoline and IS extracted from the dog plasma. Representative chromatograms of blank plasma and blank plasma spiked with both arecoline and IS are shown in Fig. 3.
3.3. Sample preparation Since arecoline has a higher recovery than arecoline hydrobromide, AgNO3 was added to react with arecoline hydrobromide, which would produce free arecoline for final quantitative analysis. The precipitate AgBr was then removed, and NaOH was added to
3.4.2. Linearity and LLOQ A calibration curve was constructed from plasma standards at six arecoline concentrations, ranging from 2 to 500 ng/mL. The ratio of peak areas of arecoline to that of IS was used for quantification. The calibration model was selected based on analysis of the
16
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
Fig. 3. Chromatograms for (A) blank plasma and (B) arecoline (a) and IS (b) in dog plasma.
data by linear regression with intercepts and a 1/×2 weighting factor. A typical equation for the arecoline calibration curve was: y = 0.0043x + 0.0361, (r2 = 0.9993), where y is the peak-area ratio of arecoline to IS and x is the plasma concentration of arecoline. The calibration curve is shown in Fig. 4. The LLOQ for arecoline was established to be 2 ng/mL, with an accuracy of 98.8%. The LOD was found to be 0.2 ng/mL arecoline. 3.4.3. Accuracy and precision The intra- and inter-day precision and accuracy for the QC samples (2, 5, 50, and 500 ng/mL) are summarized in Table 1. These data demonstrated that the developed method has satisfactory accuracy, precision, and reproducibility for the quantification of arecoline concentration in plasma.
3.4.4. Recovery and matrix effect The mean extraction recoveries of arecoline were 91.0 ± 2.8%, 94.8 ± 2.4%, and 95.5 ± 2.3% at concentrations of 5, 50, and 500 ng/mL, respectively. The mean extraction recovery of IS was 95.6 ± 2.4% at 100 ng/mL. These results suggest that the recovery of arecoline was consistent and not concentration-dependent. Recovery values are listed in Table 2. The matrix effects ranged from 92.8 ± 2.8% to 97.4 ± 2.0% for arecoline at concentrations of 5, 50, and 500 ng/mL, whereas the matrix effect for IS (100 ng/mL) was 92.5 ± 2.2%. Therefore, limited matrix effects were observed. 3.4.5. Stability The stability of arecoline was tested under freeze/thaw, shortterm, autosampler, and long-term conditions (Table 3). No stability issues were observed in any of the experiments. Arecoline was stable in dog plasma for three freeze/thaw cycles, at room temperature for 48 h, in the autosampler tray at 4 ◦ C for 48 h, and when Table 2 Recovery of arecoline (n = 6) from dog plasma. Spiked concentration (ng/mL)
5 50 500
Fig. 4. Calibration curve.
Recovery (%, n = 6)a Mean ± SD
RSD (%)b
91.0 ± 2.8 94.8 ± 2.4 95.5 ± 2.3
3.1 2.5 2.4
a Recovery = ratio of response of spiked standard before extraction to that after extraction. b RSD, relative standard deviation.
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18 Table 3 Stability of arecoline in dog plasma samples under various conditions (n = 6). Storage conditions At room temperature for 48 h At 4 ◦ C for 1 month
At -20 ◦ C for 30 days
At 4 ◦ C in the autosampler for 48 h 3 freeze-thaw cycles
Concentration (%) (ng/mL)
Accuracy (%) ±SD
5 50 500 5 50 500 5 50 500 5 50 500 5 50 500
103.5 103.1 97.2 93.6 96.7 96.4 94.1 95.5 95.9 92.7 97.6 96.3 91.3 95.6 95.8
Table 4 Pharmacokinetic parameters of arecoline after oral administration (n = 12). RSD
Parameter a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.9 2.3 2.1 2.6 1.8 2.1 2.0 2.2 1.5 2.2 2.3 1.9 2.8 3.0 2.1
17
1.8 2.2 2.2 2.8 1.9 2.2 2.1 2.3 1.6 2.4 2.4 2.0 3.1 3.1 2.2
AUC0–t (min ng/mL) AUC0–∞ (min ng/mL) b K (1/min) c Ka (1/min) d Cmax (ng/mL) e Tmax (min) f CL/F (L/min/kg) g t1/2 (min) h t1/2a (min) a b c d e f g h
Mean 15,116.86 15,771.37 0.01 0.014 60.61 120.07 0.19 69.32 49.93
CV% 19.96 18.24 13.36 11.98 0.01 0.01 15.38 9.7 7.69
AUC, area under the concentration–time curve. K, elimination rate constant. Ka , absorption rate constant. Cmax , peak plasma concentration. Tmax , peak time. CL/F , plasma clearance. t1/2 , half life. t1/2a , absorption half life.
−20 ◦ C
stored at for 30 days. Arecoline was also stable in stock solutions at room temperature for 48 h and at 4 ◦ C for 1 month. Based on these results, we concluded that arecoline can be stored and processed under routine laboratory conditions without special considerations. 3.5. Application to pharmacokinetic study The present method was successfully validated and then applied to quantitate arecoline in plasma samples after oral administration of arecoline hydrobromide tablets to beagle at a dose of 3 mg/kg. No previous studies of arecoline hydrobromide concentration in plasma samples or related pharmacokinetic studies had been reported. In our study, all samples were available and could be analyzed. The sensitivity and specificity of the assay were found to be sufficient for accurately characterizing the plasma pharmacokinetics of arecoline in dogs. After oral administration of the preparation, close and continuous visual monitoring of the animals revealed that there were no side effects. Fig. 5 shows the mean plasma concentration vs. time profile for arecoline hydrobromide in dogs after oral administration of arecoline hydrobromide tablets. The major pharmacokinetic parameters were calculated by use of a one-compartment model with first order absorption and are listed in Table 4. Tmax and Cmax were 120.07 min and 60.61 ng/mL, respectively for arecoline, and the t1/2 of arecoline was 69.32 min. The area under the concentration–time curve values, AUC0–t and AUC0–∞ , were 15,116.86 min ng/mL and 15,771.37 min ng/mL, respectively, and the CL/F of arecoline was 0.19 L/min/kg. These results indicated that arecoline was rapidly absorbed and eliminated from the blood of the dogs (Fig. 5). No significant differences were found between
male and female dogs (data not shown). This study provided useful information to serve as a basis for further research on treatments using arecoline hydrobromide tablets. Method development and evaluation of the pharmacokinetic properties of this formulation will aid the preparation of new formulations of similar drugs with improved pharmacokinetic profiles. 4. Conclusions Pharmacokinetic analysis of arecoline hydrobromide tablets requires a highly sensitive assay that can determine the arecoline concentration in plasma after oral administration. Limited volumes of plasma available and interference from the biological matrix add to the complexity of the trace analysis of arecoline. In this study, a rapid and sensitive LC–MS/MS method was developed and validated, and then successfully applied to evaluate the pharmacokinetic parameters of arecoline after oral administration of arecoline hydrobromide tablets to dog. Sample preparation is simple and relatively quick, and the analysis requires only 100 L of plasma and a short run time of less than 3 min, which is very advantageous in a pharmacokinetic study. The method has excellent sensitivity, linearity, precision, and accuracy. Currently, no bulk drug or other formulations of arecoline hydrobromide are available for clinical use. The proposed LC–MS/MS assay is an excellent technique for further evaluating the pharmacokinetic properties and therapeutic potential of the new preparation of arecoline hydrobromide tablets as a taenifuge. This study provided helpful references for the future clinical application of this herb. Acknowledgments This work was economically supported by the earmarked fund of the China Agriculture Research System (cars-38) and the Special Fund for Agro-scientific Research in the Public Interest (No. 201303038-4). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.07.043. References
Fig. 5. Mean plasma concentration–time profile after oral administration of arecoline hydrobromide tablets to dogs (n = 12) at 3 mg/kg.
[1] G.M. Simanjuntak, S.S. Margono, M. Okamoto, A. Ito, Parasitol. Today 13 (1997) 321.
18
B. Li et al. / J. Chromatogr. B 969 (2014) 12–18
[2] P.M. Schantz, P.P. Wilkins, V.C.W. Tsange, Emerging Infections 2, ASM Press, Washington, DC, 1998. [3] A. Ito, Parasitol. Int. 55 (Suppl) (2006) S3. [4] A. Ito, M. Nakao, T. Wandra, Lancet 362 (2003) 1918. [5] A. Ito, P.S. Craig, P.M. Schantz, Parasitol. Int. 55 (Suppl) (2006) S1. [6] S. Lahmar, M. Kilani, P.R. Torgerson, Ann. Trop. Med. Parasitol. 95 (2001) 69. [7] S.H. Xiao, Acta Trop. 96 (2005) 153. [8] B.M. Al-Hadiya, Profiles Drug Subst. Excip. Relat. Methodol. 32 (2005) 67. [9] F.C. Abreu, M.O. Goulart, A.M. Brett, Biosens. Bioelectron. 17 (2002) 913. [10] Z. Cai, Y. Li, L. Li, Z. Chen, J. Pharm. Anal. 2 (2012) 356. [11] J.C. He, Z.C. Dong, J.R. Wang, Chin. Arch. Tradit. Chin. Med. 29 (2011) 1967. [12] C.Y. Zhao, J.Y. Zhang, X.Z. Zhou, Y.M. Wei, J.Y. Li, Z.F. Zhang, J.S. Li, X.J. Wei, J.R. Niu, J. Tradit. Chin. Vet. Med. 26 (2007) 33. [13] Z.F. Zhang, Chinese Academy of Agricultural Sciences, Beijing, 2007.
[14] X.Z. Zhou, J.Y. Zhang, C.Y. Zhao, J.S. Li, Z.F. Zhang, J.Y. Li, X.J. Wei, J.R. Niu, L. Wang, Progr. Vet. Med. 28 (2007) 34. [15] X.Z. Zhou, J.Y. Zhang, J.S. Li, Heilongjiang Anim. Sci. Vet. Med. 21 (2012) 114. [16] X.Z. Zhou, J.Y. Zhang, C.Y. Zhao, Chin. J. Vet. Med. 41 (2007) 28. [17] X.Z. Zhou, J.Y. Zhang, J.Y. Li, J. Anhui Agric. Sci. 38 (2010) 13119, 13517. [18] H.A. Chen, Y.H. Lai, X.Y. Wang, W.R. Yang, J. Chin. Med. Mater. 25 (2002) 27. [19] W. Yuan, J.D. Lu, X.Y. Fu, Chin. J. Anal. Chem. 28 (2000) 749. [20] S. Cox, I. Piatkov, E.R. Vickers, G. Ma, J. Chromatogr. A 1032 (2004) 93. [21] J. Huang, M. McLeish, J. Chromatogr. 475 (1989) 447. [22] F.L. Ding, S.L. Peng, Acad. Period. Farm Prod. Process. 4 (2008) 77. [23] D. Pabbisetty, A. Illendula, K.M. Muraleedharan, A.G. Chittiboyina, J.S. Williamsonb, M.A. Avery, B.A. Avery, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 889–890 (2012) 125. [24] H. Fan, R. Li, Y. Gu, D. Si, C. Liu, J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 889–890 (2012) 105.
