- Email: [email protected]
A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans-ε-viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice
Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans--viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice Jiseon Kim a , Jee Sun Min a , Doyun Kim a , Yu Fen Zheng a , Karabasappa Mailar b , Won Jun Choi b , Choongho Lee b , Soo Kyung Bae a,∗ a b
College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon 14662, Republic of Korea College of Pharmacy, Dongguk University, Goyang 10326, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 June 2016 Received in revised form 22 November 2016 Accepted 23 November 2016 Available online 24 November 2016 Keywords: Trans--viniferin LC–MS/MS Mouse plasma Pharmacokinetics
a b s t r a c t In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantification of trans--viniferin in small volumes (10 l) of mouse plasma using chlorpropamide as an internal standard was developed and validated. Plasma samples were precipitated with acetonitrile and separated using an Eclipse Plus C18 column (100 × 4.6 mm, 1.8-m) with a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water (60:40 v/v) at a flow rate of 0.5 ml/min. A triple quadrupole mass spectrometer operating in positive ion mode with selected reaction-monitoring mode was used to determine trans--viniferin and chlorpropamide transitions of 455.10 → 215.05 and 277.00 → 111.00, respectively. The lower limit of quantification was 5 ng/ml with a linear range of 5–2500 ng/ml (r ≥ 0.9949). All validation data, including the selectivity, precision, accuracy, recovery, dilution integrity, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of trans--viniferin following intravenous (2.5 mg/kg), intraperitoneal (2.5, 5 and 10 mg/kg), and oral (40 mg/kg) administration in mice. This is the first report on the pharmacokinetic properties of trans-viniferin. The results provide a meaningful basis for evaluating the pre-clinical or clinical applications of trans--viniferin. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Trans--viniferin (Fig. 1), a resveratrol dehydrodimer, was first found in the common grape vine (Vitis vinifera L.) in 1977 [1], and has since been demonstrated to have antioxidant [2–4], anti-depressant [5], anti-carcinogenic [6–9], hepatoprotective [10], neuroprotective [11,12], and cardioprotective [13,14] effects. Thus, the diverse range of biological activities of trans--viniferin are of significant interest for drug research and development, and may
Abbreviations: AUC0-t , total area under the plasma concentration−time curve from time zero to time t; AUC0-∞ , total area under the plasma concentration−time curve from time zero to time infinity; F, bioavailability; Cl, total plasma clearance; Cmax , peak plasma concentration; IS, internal standard; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantitation; QC, quality control; RE, relative error; RSD, relative standard deviation; Vd, steady-state volume of distribution; t1/2 , terminal half-life; Tmax , time to reach Cmax . ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S.K. Bae). http://dx.doi.org/10.1016/j.jpba.2016.11.044 0731-7085/© 2016 Elsevier B.V. All rights reserved.
yield promising therapeutic agents. It is well known that pharmacokinetic studies play a pivotal role in drug development, as they assist in predicting a variety of efficacy- and toxicity-related events, which correlate to systemic concentrations and exposure times. However, to the best of our knowledge, the in vivo pharmacokinetic properties and oral bioavailability of trans--viniferin have not yet been assessed. Therefore, it is necessary to develop analytical methods for quantification of trans--viniferin in biological matrices. Although two LC–MS/MS methods to quantify trans-␦-viniferin, an isomer of trans--viniferin, were recently published in rat plasma [15,16], the drug isomerisom is relevant to diverse aspects of toxicology and pharmacology as isomers can produce different pharmacodynamic and pharmacokinetic properties [17]. Furthermore, these methods require 100 l of rat plasma, which are not suitable for the serial blood sampling in mice pharmacokinetic evaluation. Mice are one of the most common animal models for preclinical efficacy and pharmacokinetic assessment, particularly with limiting drug supply or specialized animal models, in early
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
117
new drug discovery stage [18]. In mice pharmacokinetic studies, small plasma volume requirements support a serial blood sampling and enable entire pharmacokinetics from a single mouse which significantly reduces the numbers of mice used and inaccuracy of the pharmacokinetics because of individual differences [18]. In this study, we report the development and validation of a simple and sensitive LC–MS/MS method to quantitate trans-viniferin in small volumes of mouse plasma for the first time. This method was successfully applied to pharmacokinetic studies of trans--viniferin in mice after intravenous, intraperitoneal, or oral administration. 2. Materials and methods 2.1. Chemicals and reagents
Fig. 1. Product ion mass spectra of trans--viniferin (A) and chlorpropamide (IS; B) with [M+H]+ at m/z 455.10 and 277.00 as the precursor ions.
Trans--viniferin (purity ≥ 95.1%; Fig. 1) was synthesized by the Medicinal Chemistry Department (Dongguk University, Goyang, Republic of Korea) based on a previously published report [19]. The chemical structure was confirmed by 1 H NMR, 13 C NMR, and high-resolution mass spectrometry. Chlorpropamide (purity ≥ 97%; Fig. 1), which was used as an internal standard (IS), dimethylsulfoxide, and formic acid were purchased from SigmaAldrich (St. Louis, MO). HPLC-grade acetonitrile and methanol were obtained from Burdick & Jackson Company (Morristown, NJ). Deionized water was prepared using the Milli-Q Plus Ultrapure Water System (Millipore Corporation, Bedford, MA). All other chemicals used were of the highest quality available. Heparinized micro-hematocrit capillary tubes were products of Thermo Fisher Scientific Inc. (Hampton, NH). Fresh heparinized mouse plasma (blank plasma) was obtained from male ICR mice (8 weeks; Orient Bio, Sungnam, Republic of Korea) and stored at −20 ◦ C prior to use. 2.2. LC–MS/MS conditions Analysis was performed using a Shimadzu Nexera X2 UHPLCLCMS 8050 system (Shimadzu Corporation, Kyoto, Japan) equipped with electrospray ionization operated in the positive ion mode. The analytes were separated by an Eclipse Plus C18 column (100 × 4.6mm, 1.8-m; Agilent, Santa Clara, CA) with an mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water (60:40, v/v) at a flow rate of 0.5 ml/min. The column and auto-sampler temperatures were maintained at 40 ◦ C and 4 ◦ C, respectively. Source dependent parameters were set as follows: nebulizing gas flow 5 l/min, drying gas flow 10 l/min, interface voltage 4.5 kV, detector voltage 1.72 kV, collision-induced dissociation argon gas pressure 230 kPa, desolvation line temperature 200 ◦ C, and heat block temperature 500 ◦ C. The optimum values for the compound dependent parameters Q1 pre bias, collision energy, and Q3 pre bias were set at 27 V, 22 V, and 22 V for trans--viniferin and 17 V, 20 V, and 15 V for the IS. The two selected reaction-monitoring (SRM) transitions per analyte (one for quantitation and the other for confirmation) were monitored for each analyte with a dwell time of 100 ms as follows: trans--viniferin, m/z 455.10 to 215.05 (quantitation) and 414.15 (confirmation); and IS m/z 277.00 to 111.00 (quantitation) and 174.96 (confirmation). LabSolutions LCMS software (version 5.6, Shimadzu) was used for analysis. 2.3. Stock solution, calibration standards, and quality controls
Fig. 2. Representative LC–MS/MS chromatograms of trans--viniferin (I) and chlorpropamide (IS) (II) in mouse plasma: (A) blank plasma, (B) blank plasma spiked with trans--viniferin (5 ng/ml) and the IS (1 g/ml), and (C) an in vivo plasma sample obtained at 120 min after intraperitoneal injection of 2.5 mg/kg trans--viniferin.
Stock solutions of 0.5 mg/ml trans--viniferin and chlorpropamide (IS) in acetonitrile were prepared. The IS stock solution was further diluted to 1 g/ml in acetonitrile for routine use. Trans--viniferin working solutions were prepared by appropriate dilution of the stock solution in acetonitrile. Calibration standards
118
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
Fig. 3. Mean plasma concentration−time curves of trans--viniferin in mice receiving (A) intravenous dose of 2.5 mg/kg (䊉, n = 8), (B) intraperitoneal doses of 2.5 mg/kg (, n = 8), 5 mg/kg (, n = 8), and 10 mg/kg (䊐, n = 8), respectively, and (C) oral dose of 40 mg/kg (䊉, n = 8). Vertical bars represent standard deviation.
Table 1 Precision, accuracy, extraction recovery and matrix effect of this assay for trans--viniferin quantification in mice plasma. Spiked (ng/mL)
5 15 300 2000
Precision (RSD, %)
Accuracy (RE, %)
Extraction recovery (n = 6)
Matrix effects (n = 8)
Intra-day (n = 6)
Inter-day (n = 5)
Intra-day (n = 6)
Inter-day (n = 5)
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
9.03 8.32 4.14 2.33
9.94 6.66 4.51 4.16
1.30 −5.43 4.83 7.93
5.16 4.34 7.52 6.96
– 95.3 ± 4.41 98.8 ± 3.99 100 ± 4.36
– 4.63 4.04 4.34
– 105 ± 4.05 94.2 ± 1.28 93.0 ± 6.29
– 3.86 1.36 6.76
Table 2 Stability of trans--viniferin in mice plasma under various conditions (n = 6). Storage conditions
Concentration (ng/ml)
Ambient temperature for 7 h
Three freeze-thaw cycles
Autosampler at 4 ◦ C for 24 h
−80 ◦ C for 90 days
Spiked
Measured (mean ± SD)
15 300 2000 15 300 2000 15 300 2000 15 300 2000
14.2 ± 0.467 279 ± 8.36 1880 ± 73.5 14.4 ± 0.968 277 ± 5.72 1860 ± 58.4 14.9 ± 1.23 282 ± 8.64 1880 ± 34.8 15.5 ± 1.21 303 ± 28.6 2090 ± 156
Precision (RSD, %)
Accuracy (RE, %)
3.30 3.00 3.90 6.71 2.06 3.14 8.24 3.06 1.85 7.84 9.44 7.50
−5.63 −6.95 −5.83 −3.80 −7.53 −6.93 −0.633 −5.85 −5.88 3.09 0.950 4.32
Table 3 Pharmacokinetic parameters (mean ± standard deviation) of trans--viniferin after intravenous, intraperitoneal, or oral administration to mice. Parameters
AUC0–t AUC0–∞ Dose-normalized AUC0–∞ t1/2 Cl Vd Cmax Dose-normalized Cmax Tmax a F
Unit
g min/ml g min/ml g min/ml min ml/min/kg ml/kg ng/ml ng/ml min (%)
Intravenous (mg/kg)
Intraperitoneal (mg/kg)
2.5
2.5
5
10
40
53.9 ± 5.13 54.8 ± 5.16 54.8 ± 5.16 115 ± 6.02 45.9 ± 4.50 1360 ± 190 – – – –
49.9 ± 8.68 51.1 ± 8.54 51.1 ± 8.54 126 ± 13.6 – – 729 ± 190 729 ± 190 15 (5–30) 93.3
92.3 ± 14.1 94.6 ± 15.3 47.3 ± 7.64 124 ± 13.0 – – 1110 ±121 554 ±60.3 15 (5–30) 86.3
194 ± 20.8 206 ± 21.1 50.4 ± 5.28 138 ± 18.4 – – 1580 ± 303 395 ± 75.6* 15 (5–30) 91.9
5.14 ± 1.87 6.76 ± 2.35 0.422 ± 0.147 204 ± 74.1 – – 42.0 ± 11.9 2.62 ± 0.741 15 (5–30) 0.771
Dose-normalized (based on 2.5 mg/kg) AUC0–∞ and Cmax values were compared when statistical analysis was performed. * 10 mg/kg group was significantly different (p < 0.05) from 2.5 mg/kg group. a Median (ranges).
Oral (mg/kg)
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
were prepared by spiking 0.5 l of appropriate working solutions with 9.5 l drug-free mouse plasma. The final concentrations used for the trans--viniferin calibration curve were 5, 10, 50, 200, 500, 1000 and 2500 ng/ml. Similarly, to assess the accuracy and precision of the assay method, quality control (QC) samples were prepared at four concentration levels: 5 (lower limit of quantification; LLOQ), 15 (low QC), 300 (medium QC), and 2000 (high QC) ng/ml. All working solutions for QC samples were prepared independently from the calibration standards. All stock solutions, working solutions, calibration standards, and QC samples were stored at −80 ◦ C. 2.4. Sample preparation A 50 l aliquot of acetonitrile containing 1 g/ml chlorpropamide was added to a 10 l aliquot of mouse plasma sample and vortexed. The mixture was then centrifuged at 13,000 × g for 15 min at 4 ◦ C to isolate and extract the supernatant. A 5 l aliquot of the supernatant was then injected into the LC–MS/MS system. All prepared samples were kept in an autosampler at 4 ◦ C until injection. 2.5. Method validation The validation parameters examined were specificity, linearity, precision, accuracy, dilution integrity, recovery, matrix effects, and stability of trans--viniferin in mouse plasma, in accordance with the US Food and Drug Administration (US FDA) guidance for the validation of bioanalytical methods [20]. The detailed procedures and their acceptance criteria are summarized in Appendix-Supplementary material. 2.6. Pharmacokinetic studies in mice Animal study protocols were approved by the Institutional Animal Care and Use Committee of the The Catholic University of Korea (Bucheon, Republic of Korea). Male ICR mice (8 weeks, 30–35 g) were purchased from Orient Bio, which were into six groups of eight mice each after a week of acclimation. Group 1 was injected trans--viniferin at a dose of 2.5 mg/kg the tail vein (0.15 ml); groups 2–4 were treated intraperitoneally (0.2 ml) at doses of 2.5, 5, 10 mg/kg, respectively; and groups 5 and 6 were given orally (0.2 ml) at doses of 10 and 40 mg/kg by oral gavage following an overnight 14 h fast. The vehicle used was dimethylsulfoxide and normal saline (1:99 v/v) for all routes of administration. Blood samples (approximately 20–30 l) were serially collected into heparinized micro-hematocrit capillary tubes (Thermo Fisher Scientific Inc.) after a slight incision of the lateral tail vein as described previously [18] at 2, 5, 15, 30, 60, 120, 240, 360, and 480 min after intravenous, and at 5, 15, 30, (45), 60, 120, 240, 360, 480, and 600 min after intraperitoneal (oral) dosing. Typically, samples for 6–7 time points were withdrawn from an individual mouse, and six separate mice were used for sample collection and analysis at each time point. The blood samples were transferred into a 0.2 ml eppendorf tube and immediately centrifuged at 15,928 × g for 5 min at 4 ◦ C to separate plasma (10 l), which were stored at −80 ◦ C until analysis. Pharmacokinetic parameters of trans--viniferin were analyzed using a non-compartmental method using WinNonlin Professional (version 2.1; Certara, Princeton, NJ). The Cmax and Tmax values were recorded directly from experimental observations. The oral (intraperitoneal) bioavailability was estimated by taking the ratio of dose-normalized AUC values after oral (intraperitoneal) doses to those after intravenous dosing. The significance of difference was assessed by one-way variance analysis (ANOVA) among three groups with Duncan’s post-hoc
119
comparison using SPSS software (version 15.0, IBM Corporation, Armonk, NY). A p value < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Optimization of LC–MS/MS conditions In order to optimize electrospray ionization for trans--viniferin and the IS, MS full scans were carried out in both positive and negative ion detection modes. Although the two analytes can be ionized in both positive and negative modes, the higher response was achieved in the positive mode. Thus, [M + H]+ ions at m/z 455.10 for trans--viniferin and 277.00 for IS were employed for method validation. The maximal fragment ions at m/z 215.05 and 111.00 were selected in SRM mode for trans--viniferin and the IS respectively to quantify trans--viniferin in mice plasma. Fig. 1 displays the product ion mass spectra of trans--viniferin and the IS at their optimal collision energies. To optimize chromatographic conditions, several types of columns, including C18 , C8 , phenyl-hexyl, cyano, and hilic columns, as well as various mobile phase compositions and flow rates, were evaluated for peak symmetry with better responses in a short analytical run time. Symmetric peak shapes with acceptable signal-to-noise ratio and adequate retention time (4 min per sample) were achieved with an Agilent Eclipse Plus C18 column (100 × 4.6-mm, 1.8-m) for trans--viniferin and the IS. Both methanol- and acetonitrile-based solutions with water and other buffers/modifiers, such as formic acid, ammonium formate, and acetic acid, were investigated as candidates for the mobile phase. Acetonitrile, rather than methanol, was chosen because of higher responses and lower background noise for trans--viniferin. The various other buffers showed no obvious advantage over water. The addition of 0.1% formic acid in an acetonitrile-water solution was found to be essential for improving the peak shapes and promoting the ionization for both analytes. As such, a mobile phase consisting of acetonitrile with 0.1% formic acid and water with 0.1% formic acid (60:40, v/v) at a flow rate of 0.5 ml/min was ultimately used. Due to lack of a stably labeled internal standard for trans-viniferin, we investigated several compounds to find a suitable IS for this assay. In this study, chlorpropamide was selected as the IS because of its robustness, stability, absence of matrix effects, and reproducible extraction. Acetonitrile-mediated protein precipitation was selected as the plasma sample preparation method because it would not require evaporation and reconstitution, significantly simplifying the sample preparation, while also ensuring the high extraction efficiency and low matrix effects required by this assay. 3.2. Method validation No obvious endogenous interference from the six batches of mouse plasma were observed at the retention times of trans-viniferin (2.01 min) and the IS (3.0 min). Fig. 2 presents typical LC–MS/MS chromatograms of blank mouse plasma (Fig. 2A), blank plasma spiked with trans--viniferin (5 ng/ml) and the IS (Fig. 2B), and an in vivo plasma sample obtained at 120 min after intraperitoneal injection of trans--viniferin (Fig. 2C). Calibration curve linearity for trans--viniferin was determined using a 1/x2 linear regression over the concentration range 5–2500 ng/ml. Correlation coefficients greater than or equal to 0.9949 for all calibration curves used in this validation (n = 6) were obtained. The typical equation for trans--viniferin calibration curves was y = 0.00833 × x − 0.00200; where y is the analyte:IS peak area ratio and x is the analyte concentration. The back-calculated
120
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
results for all calibration standards were ≤8.91% RSD and −3.89% to 0.870% RE. The LLOQ of trans--viniferin was 5 ng/ml, which presented a signal-to-noise ratio of above 10 with an acceptable accuracy and precision (within ±20%). The sensitivity of the present method was sufficient to allow pharmacokinetic studies of trans--viniferin after intravenous, intraperitoneal, or oral administration to mice. None of the analytes showed any significant peak (defined as ≥20% of the LLOQ and 5% of the IS) in blank samples injected after three repeated injections of upper limit of quantification (ULOQ) samples, indicating the absence of any carryover effects on samples. The intra- and inter-day precision and accuracy of trans-viniferin in mice plasma were all within the acceptable range of ±15% [20]. These results are summarized in Table 1, and indicate that the herein developed method was precise and accurate for the quantification of trans--viniferin in mice plasma over the established concentration ranges. The mean extraction recoveries of trans--viniferin (n = 6) at the three QC levels, 15, 300 and 2000 ng/ml, were 95.3%, 98.8%, and 100% with RSDs of 4.63, 4.04, and 4.34%, respectively (Table 1). The extraction recovery of the IS was 99.1 ± 4.21% and the RSD was 4.25% (data not shown). The matrix effects of trans--viniferin (n = 8) ranged from 93.0% to 105% with ≤ 6.76% RSD (Table 1). In addition, the matrix effects of the IS was 98.9 ± 2.63% and the RSD was 2.66% (data not shown). With a low matrix effect and highly reproducible recovery, this assay has proved to be reliable for bioanalysis. The mean accuracy (RE) of the five- and ten-fold diluted plasma samples were 1.19% and −6.80% respectively, and the precision (RSD) was 4.11% and 3.94% respectively. All values were within acceptance criteria [20]. These results suggest that samples with concentrations exceeding the ULOQ can be measured reliably when diluted appropriately. Results from short-term, three freeze–thaw cycles, posttreatment storage, and long-term storage treatments indicated that plasma trans--viniferin was stable under all storage conditions described, as the percentage deviation in concentration was within ±15% of nominal values (Table 2). The results indicated the applicability of this analytical method for routine bioanalysis. Stock solutions of trans--viniferin were also found to be stable for 20 days at 4 ◦ C and 90 days at −80 ◦ C; stored samples retained more than 98.1% of concentration compared to freshly prepared samples (data not shown). 3.3. Pharmacokinetic studies in mice The validated bioanalytical method was successfully applied to determine trans--viniferin plasma concentrations in mice following intravenous (2.5 mg/kg), intraperitoneal (2.5, 5, and 10 mg/kg), or oral (10 and 40 mg/kg) dosing. Plasma concentration–time profiles of trans--viniferin are shown in Fig. 3(A–C) and relevant pharmacokinetic parameters are listed in Table 3. Following intravenous administration of trans--viniferin, the total plasma clearance (Cl) and the steady-state volume of distribution (Vd ) were 45.9 ± 4.50 ml/min/kg and 1360 ± 190 ml/kg respectively, with an elimination half-life (t1/2 ) of 115 ± 6.02 min (Table 3). At oral dosing of 10 mg/kg, plasma concentrations of trans--viniferin obtained from only two or three blood sampling points (5–30 min), thus, the pharmacokinetic parameters of trans--viniferin could not be calculated. Following oral dosing of 40 mg/kg, the mean Cmax of trans--viniferin was 42.0 ± 11.9 ng/ml occurring at Tmax of 15 min (ranges, 5–30 min), which indicating rapid absorption (Table 3). However, the dose-normalized (based on 2.5 mg/kg) Cmax value was significantly smaller than that at intraperitoneal dosing of 2.5 mg/kg (2.62 ± 0.741 ng/ml versus 729 ± 190 ng/ml; Table 3). The calculated oral bioavailability of
trans--viniferin was 0.771%. The extremely low oral bioavailability might be owing to poor absorption and extensive metabolism in the gut. It had been reported [21] that trans--viniferin exhibited very low permeability values (0.3 × 10−6 cm/s) in the Caco-2 transwell system. Previous studies have shown that the poor oral absorption and extensive metabolism of trans-␦-viniferin, an isomer of trans--viniferin, in rats might contribute significantly to the low oral bioavailability [15,16]. After intraperitoneal injection of 2.5, 5, or 10 mg/kg trans--viniferin, plasma concentrations also increased rapidly and all mice reached the Cmax within 30 min. Concentrations then declined, with t1/2 values of 124–138 min (Table 3). The dosenormalized (based on 2.5 mg/kg) Cmax values at a dose of 10 mg/kg were significantly smaller than that at a dose of 2.5 mg/kg; the values were 729 ± 190 ng/ml, 554 ± 60.3 ng/ml, and 395 ± 75.6 ng/ml for 2.5, 5, and 10 mg/kg, respectively (Table 3). However, The AUC0–∞ values increased in a proportional fashion with increasing trans--viniferin doses. Thus, the dose-normalized AUC0–∞ values of trans--viniferin were not significantly different among the three intraperitoneal dosages studied. Moreover, there was no significance difference in t1/2 and Tmax . These results appeared to show linear pharmacokinetic properties of trans--viniferin within the dose range of 2.5–10 mg/kg in mice. The calculated bioavailability for intraperitoneal administration of trans--viniferin was 86.3–93.3%, showing a high bioavailability and indicating that intraperitoneal injection may be a more favorable route of administration for trans--viniferin. As stated previously, no information regarding the pharmacokinetic properties of trans--viniferin had been made available, and our findings therefore represent the first such pharmacokinetic data reported. 4. Conclusions We have developed and validated a simple, sensitive, and reproducible LC–MS/MS assay for the determination of trans--viniferin in mouse plasma for the first time. The present method involves a simple protein precipitation, which allowed for efficient and reproducible trans--viniferin recovery yields. The small sample volume (10 l) requirement further supports the ability to examine the pharmacokinetic profile in mice. Acknowledgements This research was supported by the Bio & Medical Technology Development Program (2013M3A9B5075838) and the Basic Research Laboratory Program (2015R1A4A1042350) through the National Research Foundation of Korea grant funded by the Korean government. 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.jpba.2016.11.044. References [1] P. Langcake, R.J. Pryce, A new class of phytoalexins from grapevines, Experientia 33 (1977) 151–152, http://dx.doi.org/10.1007/B F021 2403 4. [2] B. Baderschneider, P. Winterhalter, Isolation and characterization of novel stilbene derivatives from Riesling wine, J. Agric. Food Chem. 48 (2000) 2681–2686, http://dx.doi.org/10.1021/jf991348k. [3] Q. Kong, X. Ren, R. Hu, X. Yin, G. Jiang, Y. Pan, Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter-current chromatography, J. Sep. Sci. 39 (2016) 2374–2379, http://dx. doi.org/10.1002/jssc.201600004. [4] C. Privat, J.P. Telo, V. Bernardes-Genisson, A. Vieira, J.P. Souchard, F. Nepveu, Antioxidant properties of trans--viniferin as compared to stilbenes derivatives in aqueous and nonaqueous media, J. Agric. Food Chem. 50 (2002) 1213–1217, http://dx.doi.org/10.1021/jf010676t.
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121 ˜ [5] M. Yánez, N. Fraiz, E. Cano, F. Orallo, (−)-Trans-epsilon-viniferin, a polyphenol present in wines, is an inhibitor of noradrenaline and 5-hydroxytryptamine uptake and of monoamine oxidase activity, Eur. J. Pharmacol. 542 (2006) 54–60, http://dx.doi.org/10.1016/j.ejphar.2006.06.005. [6] C. Billard, J.C. Izard, V. Roman, C. Kern, C. Mathiot, F. Mentz, J.P. Kolb, Comparative antiproliferative and apoptotic effects of resveratrol, epsilon-viniferin and vine-shots derived polyphenols (vineatrols) on chronic B lymphocytic leukemia cells and normal human lymphocytes, Leuk. Lymphoma 43 (2002) 1991–2002, http://dx.doi.org/10.1080/ 1042819021000015952. [7] D. Colin, A. Gimazane, G. Lizard, J.C. Izard, E. Solary, N. Latruffe, D. Delmas, Effects of resveratrol analogs on cell cycle progression, cell cycle associated proteins and 5fluoro-uracil sensitivity in human derived colon cancer cells, Int. J. Cancer 124 (2009) 2780–2788, http://dx.doi.org/10.1002/ijc.24264. [8] A.K. Marel, G. Lizard, J.C. Izard, N. Latruffe, D. Delmas, Inhibitory effects of trans-resveratrol analogs molecules on the proliferation and the cell cycle progression of human colon tumoral cells, Mol. Nutr. Food Res. 52 (2008) 538–548, http://dx.doi.org/10.1002/mnfr.200700185. [9] F. Özdemir, G. Akalın, M. S¸en, N.I. Önder, A. Is¸can, H.M. Kutlu, Z. Incesu, Towards novel anti-tumor strategies for hepatic cancer: -viniferin in combination with vincristine displays pharmacodynamic synergy at lower doses in HepG2 cells, OMICS 18 (2014) 324–334, http://dx.doi.org/10.1089/ omi.2013.0045. [10] Y. Oshima, K. Namao, A. Kamijou, S. Matsuoka, M. Nakano, K. Terao, Y. Ohizumi, Powerful hepatoprotective and hepatotoxic plant oligostilbenes, isolated from the Oriental medicinal plant Vitis coignetiae (Vitaceae), Experientia 51 (1995) 63–66, http://dx.doi.org/10.1007/B F019 6492 1. [11] P.W. Groundwater, K. Hamid, I. Ng, V.J. Tallapragada, D.E. Hibbs, J. Hanrahan, The differential effects of resveratrol and trans--viniferin on the GABA-induced current in GABAA receptor subtypes expressed in Xenopus Laevis oocytes, J. Pharm. Pharm. Sci. 18 (2015) 328–338. [12] J.Y. Kim, H.Y. Jeong, H.K. Lee, S. Kim, B.Y. Hwang, K. Bae, Y.H. Seong, Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons, Phytomedicine 19 (2012) 150–159, http://dx.doi.org/10. 1016/j.phymed.2011.06.015.
121
[13] T.M. Ngoc, T.M. Hung, P.T. Thuong, M. Na, H. Kim, D.T. Ha, B.S. Min, P.T. Minh, K. Bae, Inhibition of human low density lipoprotein and high density lipoprotein oxidation by oligostilbenes from rhubarb, Biol. Pharm. Bull. 31 (2008) 1809–1812, http://dx.doi.org/10.1248/bpb.31.1809. [14] N. Zghonda, S. Yoshida, M. Araki, M. Kusunoki, A. Mliki, A. Ghorbel, H. Miyazaki, Greater effectiveness of -viniferin in red wine than its monomer resveratrol for inhibiting vascular smooth muscle cell proliferation and migration, Biosci. Biotechnol. Biochem. 75 (2011) 1259–1267, http://dx.doi. org/10.1271/bbb.110022. [15] Q. Liu, X. Liao, J. Xu, J. Zhao, J. Luo, L. Kong, Development and validation of a sensitive and selective LC–MS/MS method for the determination of trans -veniferin, a resveratrol dehydrodimer, in rat plasma and its application to pharmacokinetics and bioavailability studies, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 958 (2014) 124–129, http://dx.doi.org/10.1016/j. jchromb.2014.03.026. [16] P. Mao, Y. Lei, T. Zhang, C. Ma, B. Jin, T. Li, Pharmacokinetics, bioavailability, metabolism and excretion of -viniferin in rats, Acta Pharm. Sin. B. 6 (2016) 243–252, http://dx.doi.org/10.1016/j.apsb.2016.03.008. [17] N. Chhabra, M.L. Aseri, D. Padmanabhan, A review of drug isomerism and its significance, Int. J. Appl. Basic Med. Res. 3 (2013) 16–18, http://dx.doi.org/10. 4103/2229-516X.112233. [18] A. Watanabe, R. Watari, K. Ogawa, R. Shimizu, Y. Tanaka, N. Takai, K. Nezasa, Y. Yamaguchi, Using improved serial blood sampling method of mice to study pharmacokinetics and drug–drug interaction, J. Pharm. Sci. 104 (2015) 955–961, http://dx.doi.org/10.1002/jps.24236. [19] C.S. Yao, M. Lin, Y.H. Wang, Synthesis of the active stilbenoids by photooxidation reaction of trans--viniferin, Chin. J. Chem. 22 (2004) 1350–1355, http://dx.doi.org/10.1002/cjoc.20040221127. [20] US Department of Health and Human Services, Food and Drug Administration, Guidance for industry: Bioanalytical method validation, 2013. http://www. fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm368107.pdf (Accessed 23 June 2016). [21] I. Willenberg, M. Michael, J. Wonik, L.C. Bartel, M.T. Empl, N.H. Schebb, Investigation of the absorption of resveratrol oligomers in the Caco-2 cellular model of intestinal absorption, Food Chem. 167 (2015) 245–250, http://dx. doi.org/10.1016/j.foodchem.2014.06.103.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Short communication
A simple and sensitive liquid chromatography–tandem mass spectrometry method for trans--viniferin quantification in mouse plasma and its application to a pharmacokinetic study in mice Jiseon Kim a , Jee Sun Min a , Doyun Kim a , Yu Fen Zheng a , Karabasappa Mailar b , Won Jun Choi b , Choongho Lee b , Soo Kyung Bae a,∗ a b
College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon 14662, Republic of Korea College of Pharmacy, Dongguk University, Goyang 10326, Republic of Korea
a r t i c l e
i n f o
Article history: Received 24 June 2016 Received in revised form 22 November 2016 Accepted 23 November 2016 Available online 24 November 2016 Keywords: Trans--viniferin LC–MS/MS Mouse plasma Pharmacokinetics
a b s t r a c t In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantification of trans--viniferin in small volumes (10 l) of mouse plasma using chlorpropamide as an internal standard was developed and validated. Plasma samples were precipitated with acetonitrile and separated using an Eclipse Plus C18 column (100 × 4.6 mm, 1.8-m) with a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water (60:40 v/v) at a flow rate of 0.5 ml/min. A triple quadrupole mass spectrometer operating in positive ion mode with selected reaction-monitoring mode was used to determine trans--viniferin and chlorpropamide transitions of 455.10 → 215.05 and 277.00 → 111.00, respectively. The lower limit of quantification was 5 ng/ml with a linear range of 5–2500 ng/ml (r ≥ 0.9949). All validation data, including the selectivity, precision, accuracy, recovery, dilution integrity, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of trans--viniferin following intravenous (2.5 mg/kg), intraperitoneal (2.5, 5 and 10 mg/kg), and oral (40 mg/kg) administration in mice. This is the first report on the pharmacokinetic properties of trans-viniferin. The results provide a meaningful basis for evaluating the pre-clinical or clinical applications of trans--viniferin. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Trans--viniferin (Fig. 1), a resveratrol dehydrodimer, was first found in the common grape vine (Vitis vinifera L.) in 1977 [1], and has since been demonstrated to have antioxidant [2–4], anti-depressant [5], anti-carcinogenic [6–9], hepatoprotective [10], neuroprotective [11,12], and cardioprotective [13,14] effects. Thus, the diverse range of biological activities of trans--viniferin are of significant interest for drug research and development, and may
Abbreviations: AUC0-t , total area under the plasma concentration−time curve from time zero to time t; AUC0-∞ , total area under the plasma concentration−time curve from time zero to time infinity; F, bioavailability; Cl, total plasma clearance; Cmax , peak plasma concentration; IS, internal standard; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantitation; QC, quality control; RE, relative error; RSD, relative standard deviation; Vd, steady-state volume of distribution; t1/2 , terminal half-life; Tmax , time to reach Cmax . ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (S.K. Bae). http://dx.doi.org/10.1016/j.jpba.2016.11.044 0731-7085/© 2016 Elsevier B.V. All rights reserved.
yield promising therapeutic agents. It is well known that pharmacokinetic studies play a pivotal role in drug development, as they assist in predicting a variety of efficacy- and toxicity-related events, which correlate to systemic concentrations and exposure times. However, to the best of our knowledge, the in vivo pharmacokinetic properties and oral bioavailability of trans--viniferin have not yet been assessed. Therefore, it is necessary to develop analytical methods for quantification of trans--viniferin in biological matrices. Although two LC–MS/MS methods to quantify trans-␦-viniferin, an isomer of trans--viniferin, were recently published in rat plasma [15,16], the drug isomerisom is relevant to diverse aspects of toxicology and pharmacology as isomers can produce different pharmacodynamic and pharmacokinetic properties [17]. Furthermore, these methods require 100 l of rat plasma, which are not suitable for the serial blood sampling in mice pharmacokinetic evaluation. Mice are one of the most common animal models for preclinical efficacy and pharmacokinetic assessment, particularly with limiting drug supply or specialized animal models, in early
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
117
new drug discovery stage [18]. In mice pharmacokinetic studies, small plasma volume requirements support a serial blood sampling and enable entire pharmacokinetics from a single mouse which significantly reduces the numbers of mice used and inaccuracy of the pharmacokinetics because of individual differences [18]. In this study, we report the development and validation of a simple and sensitive LC–MS/MS method to quantitate trans-viniferin in small volumes of mouse plasma for the first time. This method was successfully applied to pharmacokinetic studies of trans--viniferin in mice after intravenous, intraperitoneal, or oral administration. 2. Materials and methods 2.1. Chemicals and reagents
Fig. 1. Product ion mass spectra of trans--viniferin (A) and chlorpropamide (IS; B) with [M+H]+ at m/z 455.10 and 277.00 as the precursor ions.
Trans--viniferin (purity ≥ 95.1%; Fig. 1) was synthesized by the Medicinal Chemistry Department (Dongguk University, Goyang, Republic of Korea) based on a previously published report [19]. The chemical structure was confirmed by 1 H NMR, 13 C NMR, and high-resolution mass spectrometry. Chlorpropamide (purity ≥ 97%; Fig. 1), which was used as an internal standard (IS), dimethylsulfoxide, and formic acid were purchased from SigmaAldrich (St. Louis, MO). HPLC-grade acetonitrile and methanol were obtained from Burdick & Jackson Company (Morristown, NJ). Deionized water was prepared using the Milli-Q Plus Ultrapure Water System (Millipore Corporation, Bedford, MA). All other chemicals used were of the highest quality available. Heparinized micro-hematocrit capillary tubes were products of Thermo Fisher Scientific Inc. (Hampton, NH). Fresh heparinized mouse plasma (blank plasma) was obtained from male ICR mice (8 weeks; Orient Bio, Sungnam, Republic of Korea) and stored at −20 ◦ C prior to use. 2.2. LC–MS/MS conditions Analysis was performed using a Shimadzu Nexera X2 UHPLCLCMS 8050 system (Shimadzu Corporation, Kyoto, Japan) equipped with electrospray ionization operated in the positive ion mode. The analytes were separated by an Eclipse Plus C18 column (100 × 4.6mm, 1.8-m; Agilent, Santa Clara, CA) with an mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water (60:40, v/v) at a flow rate of 0.5 ml/min. The column and auto-sampler temperatures were maintained at 40 ◦ C and 4 ◦ C, respectively. Source dependent parameters were set as follows: nebulizing gas flow 5 l/min, drying gas flow 10 l/min, interface voltage 4.5 kV, detector voltage 1.72 kV, collision-induced dissociation argon gas pressure 230 kPa, desolvation line temperature 200 ◦ C, and heat block temperature 500 ◦ C. The optimum values for the compound dependent parameters Q1 pre bias, collision energy, and Q3 pre bias were set at 27 V, 22 V, and 22 V for trans--viniferin and 17 V, 20 V, and 15 V for the IS. The two selected reaction-monitoring (SRM) transitions per analyte (one for quantitation and the other for confirmation) were monitored for each analyte with a dwell time of 100 ms as follows: trans--viniferin, m/z 455.10 to 215.05 (quantitation) and 414.15 (confirmation); and IS m/z 277.00 to 111.00 (quantitation) and 174.96 (confirmation). LabSolutions LCMS software (version 5.6, Shimadzu) was used for analysis. 2.3. Stock solution, calibration standards, and quality controls
Fig. 2. Representative LC–MS/MS chromatograms of trans--viniferin (I) and chlorpropamide (IS) (II) in mouse plasma: (A) blank plasma, (B) blank plasma spiked with trans--viniferin (5 ng/ml) and the IS (1 g/ml), and (C) an in vivo plasma sample obtained at 120 min after intraperitoneal injection of 2.5 mg/kg trans--viniferin.
Stock solutions of 0.5 mg/ml trans--viniferin and chlorpropamide (IS) in acetonitrile were prepared. The IS stock solution was further diluted to 1 g/ml in acetonitrile for routine use. Trans--viniferin working solutions were prepared by appropriate dilution of the stock solution in acetonitrile. Calibration standards
118
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
Fig. 3. Mean plasma concentration−time curves of trans--viniferin in mice receiving (A) intravenous dose of 2.5 mg/kg (䊉, n = 8), (B) intraperitoneal doses of 2.5 mg/kg (, n = 8), 5 mg/kg (, n = 8), and 10 mg/kg (䊐, n = 8), respectively, and (C) oral dose of 40 mg/kg (䊉, n = 8). Vertical bars represent standard deviation.
Table 1 Precision, accuracy, extraction recovery and matrix effect of this assay for trans--viniferin quantification in mice plasma. Spiked (ng/mL)
5 15 300 2000
Precision (RSD, %)
Accuracy (RE, %)
Extraction recovery (n = 6)
Matrix effects (n = 8)
Intra-day (n = 6)
Inter-day (n = 5)
Intra-day (n = 6)
Inter-day (n = 5)
Mean ± SD (%)
RSD (%)
Mean ± SD (%)
RSD (%)
9.03 8.32 4.14 2.33
9.94 6.66 4.51 4.16
1.30 −5.43 4.83 7.93
5.16 4.34 7.52 6.96
– 95.3 ± 4.41 98.8 ± 3.99 100 ± 4.36
– 4.63 4.04 4.34
– 105 ± 4.05 94.2 ± 1.28 93.0 ± 6.29
– 3.86 1.36 6.76
Table 2 Stability of trans--viniferin in mice plasma under various conditions (n = 6). Storage conditions
Concentration (ng/ml)
Ambient temperature for 7 h
Three freeze-thaw cycles
Autosampler at 4 ◦ C for 24 h
−80 ◦ C for 90 days
Spiked
Measured (mean ± SD)
15 300 2000 15 300 2000 15 300 2000 15 300 2000
14.2 ± 0.467 279 ± 8.36 1880 ± 73.5 14.4 ± 0.968 277 ± 5.72 1860 ± 58.4 14.9 ± 1.23 282 ± 8.64 1880 ± 34.8 15.5 ± 1.21 303 ± 28.6 2090 ± 156
Precision (RSD, %)
Accuracy (RE, %)
3.30 3.00 3.90 6.71 2.06 3.14 8.24 3.06 1.85 7.84 9.44 7.50
−5.63 −6.95 −5.83 −3.80 −7.53 −6.93 −0.633 −5.85 −5.88 3.09 0.950 4.32
Table 3 Pharmacokinetic parameters (mean ± standard deviation) of trans--viniferin after intravenous, intraperitoneal, or oral administration to mice. Parameters
AUC0–t AUC0–∞ Dose-normalized AUC0–∞ t1/2 Cl Vd Cmax Dose-normalized Cmax Tmax a F
Unit
g min/ml g min/ml g min/ml min ml/min/kg ml/kg ng/ml ng/ml min (%)
Intravenous (mg/kg)
Intraperitoneal (mg/kg)
2.5
2.5
5
10
40
53.9 ± 5.13 54.8 ± 5.16 54.8 ± 5.16 115 ± 6.02 45.9 ± 4.50 1360 ± 190 – – – –
49.9 ± 8.68 51.1 ± 8.54 51.1 ± 8.54 126 ± 13.6 – – 729 ± 190 729 ± 190 15 (5–30) 93.3
92.3 ± 14.1 94.6 ± 15.3 47.3 ± 7.64 124 ± 13.0 – – 1110 ±121 554 ±60.3 15 (5–30) 86.3
194 ± 20.8 206 ± 21.1 50.4 ± 5.28 138 ± 18.4 – – 1580 ± 303 395 ± 75.6* 15 (5–30) 91.9
5.14 ± 1.87 6.76 ± 2.35 0.422 ± 0.147 204 ± 74.1 – – 42.0 ± 11.9 2.62 ± 0.741 15 (5–30) 0.771
Dose-normalized (based on 2.5 mg/kg) AUC0–∞ and Cmax values were compared when statistical analysis was performed. * 10 mg/kg group was significantly different (p < 0.05) from 2.5 mg/kg group. a Median (ranges).
Oral (mg/kg)
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
were prepared by spiking 0.5 l of appropriate working solutions with 9.5 l drug-free mouse plasma. The final concentrations used for the trans--viniferin calibration curve were 5, 10, 50, 200, 500, 1000 and 2500 ng/ml. Similarly, to assess the accuracy and precision of the assay method, quality control (QC) samples were prepared at four concentration levels: 5 (lower limit of quantification; LLOQ), 15 (low QC), 300 (medium QC), and 2000 (high QC) ng/ml. All working solutions for QC samples were prepared independently from the calibration standards. All stock solutions, working solutions, calibration standards, and QC samples were stored at −80 ◦ C. 2.4. Sample preparation A 50 l aliquot of acetonitrile containing 1 g/ml chlorpropamide was added to a 10 l aliquot of mouse plasma sample and vortexed. The mixture was then centrifuged at 13,000 × g for 15 min at 4 ◦ C to isolate and extract the supernatant. A 5 l aliquot of the supernatant was then injected into the LC–MS/MS system. All prepared samples were kept in an autosampler at 4 ◦ C until injection. 2.5. Method validation The validation parameters examined were specificity, linearity, precision, accuracy, dilution integrity, recovery, matrix effects, and stability of trans--viniferin in mouse plasma, in accordance with the US Food and Drug Administration (US FDA) guidance for the validation of bioanalytical methods [20]. The detailed procedures and their acceptance criteria are summarized in Appendix-Supplementary material. 2.6. Pharmacokinetic studies in mice Animal study protocols were approved by the Institutional Animal Care and Use Committee of the The Catholic University of Korea (Bucheon, Republic of Korea). Male ICR mice (8 weeks, 30–35 g) were purchased from Orient Bio, which were into six groups of eight mice each after a week of acclimation. Group 1 was injected trans--viniferin at a dose of 2.5 mg/kg the tail vein (0.15 ml); groups 2–4 were treated intraperitoneally (0.2 ml) at doses of 2.5, 5, 10 mg/kg, respectively; and groups 5 and 6 were given orally (0.2 ml) at doses of 10 and 40 mg/kg by oral gavage following an overnight 14 h fast. The vehicle used was dimethylsulfoxide and normal saline (1:99 v/v) for all routes of administration. Blood samples (approximately 20–30 l) were serially collected into heparinized micro-hematocrit capillary tubes (Thermo Fisher Scientific Inc.) after a slight incision of the lateral tail vein as described previously [18] at 2, 5, 15, 30, 60, 120, 240, 360, and 480 min after intravenous, and at 5, 15, 30, (45), 60, 120, 240, 360, 480, and 600 min after intraperitoneal (oral) dosing. Typically, samples for 6–7 time points were withdrawn from an individual mouse, and six separate mice were used for sample collection and analysis at each time point. The blood samples were transferred into a 0.2 ml eppendorf tube and immediately centrifuged at 15,928 × g for 5 min at 4 ◦ C to separate plasma (10 l), which were stored at −80 ◦ C until analysis. Pharmacokinetic parameters of trans--viniferin were analyzed using a non-compartmental method using WinNonlin Professional (version 2.1; Certara, Princeton, NJ). The Cmax and Tmax values were recorded directly from experimental observations. The oral (intraperitoneal) bioavailability was estimated by taking the ratio of dose-normalized AUC values after oral (intraperitoneal) doses to those after intravenous dosing. The significance of difference was assessed by one-way variance analysis (ANOVA) among three groups with Duncan’s post-hoc
119
comparison using SPSS software (version 15.0, IBM Corporation, Armonk, NY). A p value < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Optimization of LC–MS/MS conditions In order to optimize electrospray ionization for trans--viniferin and the IS, MS full scans were carried out in both positive and negative ion detection modes. Although the two analytes can be ionized in both positive and negative modes, the higher response was achieved in the positive mode. Thus, [M + H]+ ions at m/z 455.10 for trans--viniferin and 277.00 for IS were employed for method validation. The maximal fragment ions at m/z 215.05 and 111.00 were selected in SRM mode for trans--viniferin and the IS respectively to quantify trans--viniferin in mice plasma. Fig. 1 displays the product ion mass spectra of trans--viniferin and the IS at their optimal collision energies. To optimize chromatographic conditions, several types of columns, including C18 , C8 , phenyl-hexyl, cyano, and hilic columns, as well as various mobile phase compositions and flow rates, were evaluated for peak symmetry with better responses in a short analytical run time. Symmetric peak shapes with acceptable signal-to-noise ratio and adequate retention time (4 min per sample) were achieved with an Agilent Eclipse Plus C18 column (100 × 4.6-mm, 1.8-m) for trans--viniferin and the IS. Both methanol- and acetonitrile-based solutions with water and other buffers/modifiers, such as formic acid, ammonium formate, and acetic acid, were investigated as candidates for the mobile phase. Acetonitrile, rather than methanol, was chosen because of higher responses and lower background noise for trans--viniferin. The various other buffers showed no obvious advantage over water. The addition of 0.1% formic acid in an acetonitrile-water solution was found to be essential for improving the peak shapes and promoting the ionization for both analytes. As such, a mobile phase consisting of acetonitrile with 0.1% formic acid and water with 0.1% formic acid (60:40, v/v) at a flow rate of 0.5 ml/min was ultimately used. Due to lack of a stably labeled internal standard for trans-viniferin, we investigated several compounds to find a suitable IS for this assay. In this study, chlorpropamide was selected as the IS because of its robustness, stability, absence of matrix effects, and reproducible extraction. Acetonitrile-mediated protein precipitation was selected as the plasma sample preparation method because it would not require evaporation and reconstitution, significantly simplifying the sample preparation, while also ensuring the high extraction efficiency and low matrix effects required by this assay. 3.2. Method validation No obvious endogenous interference from the six batches of mouse plasma were observed at the retention times of trans-viniferin (2.01 min) and the IS (3.0 min). Fig. 2 presents typical LC–MS/MS chromatograms of blank mouse plasma (Fig. 2A), blank plasma spiked with trans--viniferin (5 ng/ml) and the IS (Fig. 2B), and an in vivo plasma sample obtained at 120 min after intraperitoneal injection of trans--viniferin (Fig. 2C). Calibration curve linearity for trans--viniferin was determined using a 1/x2 linear regression over the concentration range 5–2500 ng/ml. Correlation coefficients greater than or equal to 0.9949 for all calibration curves used in this validation (n = 6) were obtained. The typical equation for trans--viniferin calibration curves was y = 0.00833 × x − 0.00200; where y is the analyte:IS peak area ratio and x is the analyte concentration. The back-calculated
120
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121
results for all calibration standards were ≤8.91% RSD and −3.89% to 0.870% RE. The LLOQ of trans--viniferin was 5 ng/ml, which presented a signal-to-noise ratio of above 10 with an acceptable accuracy and precision (within ±20%). The sensitivity of the present method was sufficient to allow pharmacokinetic studies of trans--viniferin after intravenous, intraperitoneal, or oral administration to mice. None of the analytes showed any significant peak (defined as ≥20% of the LLOQ and 5% of the IS) in blank samples injected after three repeated injections of upper limit of quantification (ULOQ) samples, indicating the absence of any carryover effects on samples. The intra- and inter-day precision and accuracy of trans-viniferin in mice plasma were all within the acceptable range of ±15% [20]. These results are summarized in Table 1, and indicate that the herein developed method was precise and accurate for the quantification of trans--viniferin in mice plasma over the established concentration ranges. The mean extraction recoveries of trans--viniferin (n = 6) at the three QC levels, 15, 300 and 2000 ng/ml, were 95.3%, 98.8%, and 100% with RSDs of 4.63, 4.04, and 4.34%, respectively (Table 1). The extraction recovery of the IS was 99.1 ± 4.21% and the RSD was 4.25% (data not shown). The matrix effects of trans--viniferin (n = 8) ranged from 93.0% to 105% with ≤ 6.76% RSD (Table 1). In addition, the matrix effects of the IS was 98.9 ± 2.63% and the RSD was 2.66% (data not shown). With a low matrix effect and highly reproducible recovery, this assay has proved to be reliable for bioanalysis. The mean accuracy (RE) of the five- and ten-fold diluted plasma samples were 1.19% and −6.80% respectively, and the precision (RSD) was 4.11% and 3.94% respectively. All values were within acceptance criteria [20]. These results suggest that samples with concentrations exceeding the ULOQ can be measured reliably when diluted appropriately. Results from short-term, three freeze–thaw cycles, posttreatment storage, and long-term storage treatments indicated that plasma trans--viniferin was stable under all storage conditions described, as the percentage deviation in concentration was within ±15% of nominal values (Table 2). The results indicated the applicability of this analytical method for routine bioanalysis. Stock solutions of trans--viniferin were also found to be stable for 20 days at 4 ◦ C and 90 days at −80 ◦ C; stored samples retained more than 98.1% of concentration compared to freshly prepared samples (data not shown). 3.3. Pharmacokinetic studies in mice The validated bioanalytical method was successfully applied to determine trans--viniferin plasma concentrations in mice following intravenous (2.5 mg/kg), intraperitoneal (2.5, 5, and 10 mg/kg), or oral (10 and 40 mg/kg) dosing. Plasma concentration–time profiles of trans--viniferin are shown in Fig. 3(A–C) and relevant pharmacokinetic parameters are listed in Table 3. Following intravenous administration of trans--viniferin, the total plasma clearance (Cl) and the steady-state volume of distribution (Vd ) were 45.9 ± 4.50 ml/min/kg and 1360 ± 190 ml/kg respectively, with an elimination half-life (t1/2 ) of 115 ± 6.02 min (Table 3). At oral dosing of 10 mg/kg, plasma concentrations of trans--viniferin obtained from only two or three blood sampling points (5–30 min), thus, the pharmacokinetic parameters of trans--viniferin could not be calculated. Following oral dosing of 40 mg/kg, the mean Cmax of trans--viniferin was 42.0 ± 11.9 ng/ml occurring at Tmax of 15 min (ranges, 5–30 min), which indicating rapid absorption (Table 3). However, the dose-normalized (based on 2.5 mg/kg) Cmax value was significantly smaller than that at intraperitoneal dosing of 2.5 mg/kg (2.62 ± 0.741 ng/ml versus 729 ± 190 ng/ml; Table 3). The calculated oral bioavailability of
trans--viniferin was 0.771%. The extremely low oral bioavailability might be owing to poor absorption and extensive metabolism in the gut. It had been reported [21] that trans--viniferin exhibited very low permeability values (0.3 × 10−6 cm/s) in the Caco-2 transwell system. Previous studies have shown that the poor oral absorption and extensive metabolism of trans-␦-viniferin, an isomer of trans--viniferin, in rats might contribute significantly to the low oral bioavailability [15,16]. After intraperitoneal injection of 2.5, 5, or 10 mg/kg trans--viniferin, plasma concentrations also increased rapidly and all mice reached the Cmax within 30 min. Concentrations then declined, with t1/2 values of 124–138 min (Table 3). The dosenormalized (based on 2.5 mg/kg) Cmax values at a dose of 10 mg/kg were significantly smaller than that at a dose of 2.5 mg/kg; the values were 729 ± 190 ng/ml, 554 ± 60.3 ng/ml, and 395 ± 75.6 ng/ml for 2.5, 5, and 10 mg/kg, respectively (Table 3). However, The AUC0–∞ values increased in a proportional fashion with increasing trans--viniferin doses. Thus, the dose-normalized AUC0–∞ values of trans--viniferin were not significantly different among the three intraperitoneal dosages studied. Moreover, there was no significance difference in t1/2 and Tmax . These results appeared to show linear pharmacokinetic properties of trans--viniferin within the dose range of 2.5–10 mg/kg in mice. The calculated bioavailability for intraperitoneal administration of trans--viniferin was 86.3–93.3%, showing a high bioavailability and indicating that intraperitoneal injection may be a more favorable route of administration for trans--viniferin. As stated previously, no information regarding the pharmacokinetic properties of trans--viniferin had been made available, and our findings therefore represent the first such pharmacokinetic data reported. 4. Conclusions We have developed and validated a simple, sensitive, and reproducible LC–MS/MS assay for the determination of trans--viniferin in mouse plasma for the first time. The present method involves a simple protein precipitation, which allowed for efficient and reproducible trans--viniferin recovery yields. The small sample volume (10 l) requirement further supports the ability to examine the pharmacokinetic profile in mice. Acknowledgements This research was supported by the Bio & Medical Technology Development Program (2013M3A9B5075838) and the Basic Research Laboratory Program (2015R1A4A1042350) through the National Research Foundation of Korea grant funded by the Korean government. 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.jpba.2016.11.044. References [1] P. Langcake, R.J. Pryce, A new class of phytoalexins from grapevines, Experientia 33 (1977) 151–152, http://dx.doi.org/10.1007/B F021 2403 4. [2] B. Baderschneider, P. Winterhalter, Isolation and characterization of novel stilbene derivatives from Riesling wine, J. Agric. Food Chem. 48 (2000) 2681–2686, http://dx.doi.org/10.1021/jf991348k. [3] Q. Kong, X. Ren, R. Hu, X. Yin, G. Jiang, Y. Pan, Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter-current chromatography, J. Sep. Sci. 39 (2016) 2374–2379, http://dx. doi.org/10.1002/jssc.201600004. [4] C. Privat, J.P. Telo, V. Bernardes-Genisson, A. Vieira, J.P. Souchard, F. Nepveu, Antioxidant properties of trans--viniferin as compared to stilbenes derivatives in aqueous and nonaqueous media, J. Agric. Food Chem. 50 (2002) 1213–1217, http://dx.doi.org/10.1021/jf010676t.
J. Kim et al. / Journal of Pharmaceutical and Biomedical Analysis 134 (2017) 116–121 ˜ [5] M. Yánez, N. Fraiz, E. Cano, F. Orallo, (−)-Trans-epsilon-viniferin, a polyphenol present in wines, is an inhibitor of noradrenaline and 5-hydroxytryptamine uptake and of monoamine oxidase activity, Eur. J. Pharmacol. 542 (2006) 54–60, http://dx.doi.org/10.1016/j.ejphar.2006.06.005. [6] C. Billard, J.C. Izard, V. Roman, C. Kern, C. Mathiot, F. Mentz, J.P. Kolb, Comparative antiproliferative and apoptotic effects of resveratrol, epsilon-viniferin and vine-shots derived polyphenols (vineatrols) on chronic B lymphocytic leukemia cells and normal human lymphocytes, Leuk. Lymphoma 43 (2002) 1991–2002, http://dx.doi.org/10.1080/ 1042819021000015952. [7] D. Colin, A. Gimazane, G. Lizard, J.C. Izard, E. Solary, N. Latruffe, D. Delmas, Effects of resveratrol analogs on cell cycle progression, cell cycle associated proteins and 5fluoro-uracil sensitivity in human derived colon cancer cells, Int. J. Cancer 124 (2009) 2780–2788, http://dx.doi.org/10.1002/ijc.24264. [8] A.K. Marel, G. Lizard, J.C. Izard, N. Latruffe, D. Delmas, Inhibitory effects of trans-resveratrol analogs molecules on the proliferation and the cell cycle progression of human colon tumoral cells, Mol. Nutr. Food Res. 52 (2008) 538–548, http://dx.doi.org/10.1002/mnfr.200700185. [9] F. Özdemir, G. Akalın, M. S¸en, N.I. Önder, A. Is¸can, H.M. Kutlu, Z. Incesu, Towards novel anti-tumor strategies for hepatic cancer: -viniferin in combination with vincristine displays pharmacodynamic synergy at lower doses in HepG2 cells, OMICS 18 (2014) 324–334, http://dx.doi.org/10.1089/ omi.2013.0045. [10] Y. Oshima, K. Namao, A. Kamijou, S. Matsuoka, M. Nakano, K. Terao, Y. Ohizumi, Powerful hepatoprotective and hepatotoxic plant oligostilbenes, isolated from the Oriental medicinal plant Vitis coignetiae (Vitaceae), Experientia 51 (1995) 63–66, http://dx.doi.org/10.1007/B F019 6492 1. [11] P.W. Groundwater, K. Hamid, I. Ng, V.J. Tallapragada, D.E. Hibbs, J. Hanrahan, The differential effects of resveratrol and trans--viniferin on the GABA-induced current in GABAA receptor subtypes expressed in Xenopus Laevis oocytes, J. Pharm. Pharm. Sci. 18 (2015) 328–338. [12] J.Y. Kim, H.Y. Jeong, H.K. Lee, S. Kim, B.Y. Hwang, K. Bae, Y.H. Seong, Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons, Phytomedicine 19 (2012) 150–159, http://dx.doi.org/10. 1016/j.phymed.2011.06.015.
121
[13] T.M. Ngoc, T.M. Hung, P.T. Thuong, M. Na, H. Kim, D.T. Ha, B.S. Min, P.T. Minh, K. Bae, Inhibition of human low density lipoprotein and high density lipoprotein oxidation by oligostilbenes from rhubarb, Biol. Pharm. Bull. 31 (2008) 1809–1812, http://dx.doi.org/10.1248/bpb.31.1809. [14] N. Zghonda, S. Yoshida, M. Araki, M. Kusunoki, A. Mliki, A. Ghorbel, H. Miyazaki, Greater effectiveness of -viniferin in red wine than its monomer resveratrol for inhibiting vascular smooth muscle cell proliferation and migration, Biosci. Biotechnol. Biochem. 75 (2011) 1259–1267, http://dx.doi. org/10.1271/bbb.110022. [15] Q. Liu, X. Liao, J. Xu, J. Zhao, J. Luo, L. Kong, Development and validation of a sensitive and selective LC–MS/MS method for the determination of trans -veniferin, a resveratrol dehydrodimer, in rat plasma and its application to pharmacokinetics and bioavailability studies, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 958 (2014) 124–129, http://dx.doi.org/10.1016/j. jchromb.2014.03.026. [16] P. Mao, Y. Lei, T. Zhang, C. Ma, B. Jin, T. Li, Pharmacokinetics, bioavailability, metabolism and excretion of -viniferin in rats, Acta Pharm. Sin. B. 6 (2016) 243–252, http://dx.doi.org/10.1016/j.apsb.2016.03.008. [17] N. Chhabra, M.L. Aseri, D. Padmanabhan, A review of drug isomerism and its significance, Int. J. Appl. Basic Med. Res. 3 (2013) 16–18, http://dx.doi.org/10. 4103/2229-516X.112233. [18] A. Watanabe, R. Watari, K. Ogawa, R. Shimizu, Y. Tanaka, N. Takai, K. Nezasa, Y. Yamaguchi, Using improved serial blood sampling method of mice to study pharmacokinetics and drug–drug interaction, J. Pharm. Sci. 104 (2015) 955–961, http://dx.doi.org/10.1002/jps.24236. [19] C.S. Yao, M. Lin, Y.H. Wang, Synthesis of the active stilbenoids by photooxidation reaction of trans--viniferin, Chin. J. Chem. 22 (2004) 1350–1355, http://dx.doi.org/10.1002/cjoc.20040221127. [20] US Department of Health and Human Services, Food and Drug Administration, Guidance for industry: Bioanalytical method validation, 2013. http://www. fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm368107.pdf (Accessed 23 June 2016). [21] I. Willenberg, M. Michael, J. Wonik, L.C. Bartel, M.T. Empl, N.H. Schebb, Investigation of the absorption of resveratrol oligomers in the Caco-2 cellular model of intestinal absorption, Food Chem. 167 (2015) 245–250, http://dx. doi.org/10.1016/j.foodchem.2014.06.103.