J S S
ISSN 1615-9306 · JSSCCJ 38 (11) 1813–2006 (2015) · Vol. 38 · No. 11 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
11 15
www.jss-journal.com
1872 Woong-Kee Choi1 ∗ Kee Dong Yoon1 ∗ Joeng Kee Lee1 Jung Bae Park1 Tae-Hwe Heo1 Choongho Lee2 Soo Kyung Bae1 1 College
of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, South Korea 2 College of Pharmacy, Dongguk University, Goyang, South Korea Received January 19, 2015 Revised March 2, 2015 Accepted March 11, 2015
J. Sep. Sci. 2015, 38, 1872–1880
Research Article
Development and validation of a liquid chromatography with tandem mass spectrometry method for the quantification of vitisin B in rat plasma and urine A new, rapid, and sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of vitisin B and validated in rat plasma and urine using carbamazepine as an internal standard. The plasma (0.05 mL) or urine (0.2 mL) samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Eclipse Plus C18 column (100 × 4.6 mm, 3.5 m) with a mobile phase consisting of acetonitrile and 0.1% formic acid water (60:40, v/v) at a flow rate of 0.7 mL/min. Detection and quantification were performed by mass spectrometry in selected reaction-monitoring mode with positive electrospray ionization. The calibration curves were recovered over the concentration ranges of 10−5000 ng/mL (correlation coefficients, r0.9833) in plasma and 5−2500 ng/mL (r0.9977) in urine, respectively. All validation data, including the specificity, precision, accuracy, recovery, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of vitisin B following intravenous administration of 0.5 and 1 mg/kg and intraperitoneal injection of 5, 10, and 25 mg/kg to rats. This is the first report on the pharmacokinetic properties of vitisin B. The results provide a meaningful basis to evaluate preclinical or clinical applications of vitisin B. Keywords: Liquid chromatography with tandem mass spectrometry / Pharmacokinetics / Plasma / Urine / Vitisin B DOI 10.1002/jssc.201500071
1 Introduction Resveratrol oligomers are characterized by the polymerization of two to eight resveratrol units and are the largest group of oligomeric stilbenes [1, 2]. Resveratrol oligomer polyphenols were mainly isolated from five plant families; namely, Vitaceae, Leguminosae, Gnetaceae, Dipterocarpaceae, and Cyperaceae [1–5]. In addition, resveratrol oligomers were recognized as fungal detoxification products of resveratrol metabolism. These oligomers exhibit diverse biological activities, such as antibacterial, antifungal, anticancer, anti-HIV, Correspondence: Professor Soo Kyung Bae, College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do, 420–743, South Korea E-mail: [email protected] Fax: +82-2-2164-4096
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; CL, total body clearance; Cmax , peak plasma concentration; F, bioavailability; LLOQ, lower limit of quantitation; QC, quality control; RE, relative error; Tmax , time to reach Cmax ; Vdss , steady-state volume of distribution t1/2 , terminal half-life C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and antioxidant effects [6–8]. Thus, it is important to explore the beneficial effects of resveratrol oligomers. Vitisin B, a resveratrol tetramer, was found to have a variety of pharmacological effects including cardiovascular protective [9], antimicrobial [10, 11], anti-inflammatory [12, 13], antileukemic [14], and cholesterol-lowering activities [15], making it a promising candidate for further research. Although many pharmacological data on vitisin B have been published [9–15], no information is available regarding its pharmacokinetic properties in animals. Based on the clinical potential of vitisin B, it is important to understand its pharmacokinetic characteristics. To date, no analytical method for vitisin B in biological fluids has been reported. Several HPLC–UV and LC–MS methods were published to confirm the structures and the detectable amount of vitisin B in plant extracts [13, 14, 16, 17]. However, these methods cannot typically be applied directly to the bioanalysis of vitisin B owing to interference from endogenous materials, insufficient sensitivity, and long retention times. Moreover, these methods have not been validated. Therefore, a more sensitive and rapid analysis method is required for monitoring of vitisin B concentrations in biological samples.
∗ Both
authors equally contributed to this work.
www.jss-journal.com
J. Sep. Sci. 2015, 38, 1872–1880
In this study, a rapid and sensitive LC–MS/MS method to determine vitisin B in rat plasma and urine was developed, validated, and successfully applied to pharmacokinetic studies of vitisin B after intravenous administration (0.5 and 1 mg/kg) and intraperitoneal injection (5, 10, and 25 mg/kg) in rats.
2 Materials and methods 2.1 Materials and reagents Vitisin B (purity: 95.7%) was isolated from Vitis amurensis as described previously [17]. Carbamazepine, dimethylsulfoxide, formic acid, and PEG 400 were purchased from Sigma–Aldrich. Ethyl acetate was obtained from Burdick & Jackson (Morristown, NJ, USA). All solvents were of HPLC grade. All other chemicals were of the highest quality available. Freshly obtained rat plasma (blank plasma) and urine (blank urine) were collected from male Sprague–Dawley rats and stored at –20⬚C before use.
2.2 Calibration standards and QCs Stock solutions of vitisin B and carbamazepine (internal standard, IS) were prepared by dissolving in dimethylsulfoxide at 5 mg/mL, respectively. The IS stock solution was further diluted to 50 ng/mL in acetonitrile for routine use. The working solutions of vitisin B were prepared by serial dilutions of the stock solution with dimethylsulfoxide to obtain the final desired concentrations. Calibration standards and QC samples in plasma were prepared by spiking 2.5 L of appropriate working standard or QC solution with 47.5 L drug-free rat plasma. The ranges of calibration curves in rat plasma were 10, 20, 100, 200, 500, 2000, and 5000 ng/mL. Additionally, the QC samples were prepared in drug-free rat plasma at four different concentration levels of 10 (lower limit of quantification; LLOQ), 30 (low QC), 400 (medium QC), and 4000 ng/mL (high QC) to evaluate the intra- and inter-day precision and accuracy of this method. The calibration standards and QC samples in rat urine were prepared by spiking 10 L of appropriate working standard or QC solution with 190 L of drug-free rat urine. The ranges of calibration curves in rat urine were 5, 10, 50, 100, 250, 1000, and 2500 ng/mL and the QC levels in rat urine were 5 (LLOQ), 15 (low QC), 200 (medium QC), and 2000 ng/mL (high QC). The stock solutions for QC samples were prepared independently from the calibration standards. On the day of analysis, calibration curves for vitisin B in rat plasma or urine were derived from their peak area ratios of vitisin B to the IS versus the respective plasma or urine concentrations of vitisin B using a linear regression with 1/x2 as a weighting factor. The respective QC samples were assayed along with each batch of plasma or urine samples. All prepared plasma or urine samples and stock solutions were stored at –80⬚C (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC, USA). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.3 Sample preparation For plasma samples, 10 L of the IS solution containing 50 ng/mL carbamazepine were mixed with 50 L plasma sample, after which 1 mL of ethyl acetate were added and vortex-mixed for 5 min. After centrifugation (16 000 ×g for 15 min), the organic layer was transferred to another tube and was completely evaporated under a gentle stream of nitrogen gas at 40⬚C. The residue was reconstituted with 100 L of mobile phase and centrifuged (16 000 ×g for 10 min at 4⬚C). Then, a 10 L aliquot was injected into the LC–MS/MS system for analysis. For urine samples, 10 L of the IS (50 ng/mL carbamazepine) were mixed with 200 L urine sample, after which 1.5 mL of ethyl acetate were added and vortex-mixed for 5 min. Other procedures were similar to those used in the analysis of plasma samples.
2.4 LC–MS/MS conditions The samples were analyzed using a API 5500 Q-Trap mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with a 1260 HPLC system (Agilent Technologies, Wilmington, DE, USA) in ESI mode used to generate positive [M+H]+ . The compounds were separated on a C18 reversed-phase column (Eclipse Plus C18 column, 100 × 4.6 mm i.d., 3.5 m particle size; Agilent) with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (60:40, v/v) at a flow rate of 0.7 mL/min. The column and autosampler temperatures were maintained at 40 and 4⬚C, respectively. The total run time was 3.5 min for each sample. The optimized ion spray voltage and temperature were set at 5500 V and 600⬚C, respectively. The operating conditions, which were optimized by flow injection of vitisin B, were as follows: declustering potential and collision energy were 30 and 30 V for vitisin B and 190 and 25 V for the IS, respectively. The entrance potential and collision cell exit potentials were 10 and 5 V, respectively. Nitrogen gas was used as the nebulizer gas, curtain gas, and collision-activated dissociation gas, which were set at 20, 70, and 30 psi, respectively. The mass transitions used for vitisin B and the IS were m/z 907.3→559.2 and 237.2 → 194.1, respectively. Data acquisition was performed with dwell times of 150 ms, and quadrupoles Q1 and Q3 were set on unit resolution. The analytical data were processed using the Analyst software (version 1.5.2; Applied Biosystems, Foster City, CA, USA).
2.5 LC–MS/MS method validation The validation parameters were selectivity, linearity, sensitivity, accuracy, precision, and matrix effects of the assay and the recovery and stability of vitisin B in rat plasma or urine, in accordance with the US Food and Drug Administration (US FDA) guidance for the validation of bioanalytical methods [18]. www.jss-journal.com
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The specificity was assessed by comparing the chromatograms of six different batches of drug-free rat plasma or urine to ensure that no interfering peaks were present at the retention times of vitisin B(10 ng/mL for plasma and 5 ng/mL for urine) and the IS at the LLOQ. The carryover was investigated by injecting two double blank plasma or urine samples directly after a highest concentration sample from the respective calibration curves (upper limit of quantification; ULOQ) in an analytical run. The carryover test was met if no interfering peaks appeared with areas greater than 20% of the peak areas at the LLOQ level of each analyte and 5% of the IS peak area. Calibration curves were prepared by assaying standard rat plasma or urine samples of vitisin B at the concentration ranges of 10–5000 ng/mL for rat plasma and 5–2500 ng/mL for rat urine. The linearity of each method matched calibration curve was determined by plotting the peak area ratio (y) of vitisin B to the IS versus the nominal concentration (x) of vitisin B. The calibration model was selected based on analysis of data by linear regression with weighting factors (1/x, 1/x2 , and none).The LLOQ of the assay was defined as the lowest concentration on the calibration curve of an analyte with precision (±20%) and accuracy (80–120%) that could be quantitated with an S/N of at least 10. The inter-day precision and accuracy were evaluated by analyzing ten replicates of the LLOQ sample and three different QC samples on five days (two replicates per day).The intra-day precision and accuracy were determined by analyzing six replicates of the LLOQ sample and three different QC samples on the same day. The precision was determined based on the RSD (RSD,%), and the accuracy was expressed as the relative error (RE): RE (%) = (found concentration − theoretical concentration)/theoretical concentration × 100%. The concentrations of QC samples, including LLOQ samples, were determined from the standard calibration curve and were analyzed on the same day. The acceptance criterion was ±15% deviation from the normal value, except at the LLOQ (±20%). To evaluate extraction recoveries and matrix effects, six different batches of drug-free rat plasma or urine were used. Extraction recoveries of vitisin B were determined by comparing the peak areas of extracted QC samples with the peak areas of post-extraction plasma or urine blanks spiked with the corresponding concentrations at three QC levels [19]. The matrix effects on the ionization of the analytes were evaluated by comparing the peak areas of post-extraction blank plasma or urine spiked at the concentrations of the QC samples with the areas obtained by direct injection of the corresponding standard solutions [19]. The matrix effects and extraction recovery of the IS at 50 ng/mL were evaluated using the same method. All assays were performed in sextuplicate. The dilution integrity test was performed to ensure that plasma samples could be diluted with blank plasma without affecting the final concentration. The dilution QC samples (25 000 ng/mL for plasma) were diluted tenfold with blank rat plasma before extraction in six replicates and analyzed. As part of the validation, the replicates had to comply with both C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2015, 38, 1872–1880
precision of 15% and accuracy of 85–115%, similar to other QC samples. Stability tests for stock solutions of vitisin B and the IS were conducted at 4 and –80⬚C. Stability in the stock solution was evaluated by comparing the area response of the analytes (stability samples; stored) with the response of the samples prepared from a fresh stock solution. The solutions were considered stable if the deviation from the nominal value was within ±10.0%. The stabilities of vitisin B in rat plasma or urine were established at low QC and high QC levels using six replicates under the following conditions: (1) short-term storage (8 h at room temperature), (2) long-term storage (90 days at –80⬚C), (3) three freeze–thaw cycles, and (4) post-treatment storage (24 h at 4⬚C). Stability results in plasma were evaluated by measuring the area ratio response (analyte/IS) of the stability samples against freshly prepared QC samples with identical concentrations. Stability data in plasma or urine were acceptable if the deviation from the mean area ratio response of freshly prepared QC samples was within ±15.0%.
2.6 Pharmacokinetic studies in rats Pharmacokinetic studies were performed in rats to verify the applicability of the method. Male Sprague–Dawley rats (eight weeks old, 250–270 g) were purchased from Orient Bio (Sungnam, Gyeonggi-do, South Korea). The protocol for the animal study was approved by the Institutional Animal Care and Use Committee on the Sungsim Campus of The Catholic University of Korea (Bucheon, South Korea). The procedures used for housing and handling were as reported previously [20]. The jugular vein (for drug administration for intravenous study only) and carotid arteries (for blood sampling) of each rat were cannulated with a polyethylene tube (Clay Adams, Franklin Lakes, NJ, USA). Each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from aesthesia for 4–5 h before beginning the experiment. Thus, rats were not restrained in the present study. Vitisin B (dissolved in dimethylsulfoxide: PEG200: normal saline = 2:48:50, v/v) at doses of 0.5 (n = 5) and 1 (n = 5) mg/kg was infused (total infusion volume of 2 mL/kg) over 1 min by the jugular vein to rats. Approximately 0.12 mL of blood from each rat was collected by the carotid artery at 0 (control), 1 (at the end of the infusion), 5, 10, 15, 30, 45, 60, 90, and 120 min after the start of the intravenous infusion of vitisin B. A heparinized 0.9% NaCl-injectable solution (20 units/mL, 0.3 mL) was used to flush the cannula immediately after each blood sampling to prevent blood clotting. The blood samples were immediately centrifuged at 13 000 rpm for 10 min at 4⬚C and the plasma samples (50 L) were stored at –80⬚C until LC–MS/MS analysis. At the end of 24 h, each metabolic cage was rinsed with 10 mL of distilled water and the rinsings were combined with the 24 h urine sample. After measuring the exact volume of the combined urine sample, two 200 L aliquots of the combined urine sample were stored at –80⬚C until LC–MS/MS www.jss-journal.com
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analysis. After the experiments, the rats were euthanized by CO2 gas. Vitisin B (the same solution used in the intravenous study) at doses of 5 (n = 5), 10 (n = 5), and 25 mg/kg (n = 5) was injected intraperitoneally (total injection volume of 3 mL/kg). A blood sample (approximately 0.12 mL) was collected by the carotid artery at 0, 5, 15, 30, 60, 120, 180, 240, 360, 480, 600, 720, 1080, and 1440 min after intraperitoneal injection of vitisin B. Other procedures were as for the intravenous study. Pharmacokinetic parameters including the terminal halflife (t1/2 ), area under concentration–time curve (AUC0–t and AUC0– ), total body clearance (CL), and steady-state volume of distribution (Vdss ) of vitisin B were analyzed using a noncompartmental method using WinNonlin Professional, version 2.1. The maximum plasma concentration (Cmax ) and the time at which the maximum plasma concentration was obtained (Tmax ) were the observed values. The bioavailability (F) for intraperitoneal vitisin B was calculated as: F = AUCip /AUCiv × doseiv /doseip × 100%
(1)
where AUCip is the AUC0– after intraperitoneal injection of vitisin B; and AUCiv is the AUC0– after intravenous administration of vitisin B at a dose of 1 mg/kg. A p value < 0.05 was deemed to be statistically significant using a t-test between the two means for the unpaired data, or a Duncan’s multiple range test of Statistical Package for the Social Sciences (SPSS) posteriori analysis of variance (ANOVA) among the three means for the unpaired data.
3 Results and discussion 3.1 LC–MS/MS optimization To optimize the MS parameters, vitisin B stock solutions were directly injected into the ion source of the mass spectrometer using a syringe pump. In positive-ion mode, vitisin B and the IS gave deprotonated molecular ions, [M+H]+ , as the major species. Final optimization of mass parameters was performed using the automatic tuning tool to obtain maximum sensitivity of selected reaction-monitoring quantification. Figure 1 displays the product ion mass spectra of vitisin B and the IS at their optimal collision energies. To optimize chromatographic conditions, many column types, including C18 , C8 , phenyl-hexyl, cyano, and HILIC columns, and various compositions of mobile phase were evaluated for resolution, peak symmetry, and retention time. The use of an Agilent Eclipse Plus C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% aqueous formic acid (60:40, v/v) resulted in the best peak shape and highest sensitivity. Initially, protein precipitation using methanol and acetonitrile for sample preparation was attempted, but significant lower sensitivity hindered further development. Thus, a LLE procedure was investigated. Several organic solvents, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Product ion mass spectra of vitisin B (A) and carbamazepine (IS; B) with [M+H]+ at m/z 559.2 and 194.1 as the precursor ions.
including ethyl acetate, ether, dichloromethane, acetone, chloroform, methyl tert-butyl ether, and their mixtures were evaluated. Finally, ethyl acetate was found to be optimal, producing a clean chromatogram for blank plasma or urine samples, constant extraction recovery, and negligible matrix effects. For a natural product with a complex molecular structure, isotope-labeled IS was difficult to obtain. It was challenging to select an appropriate IS with a similar retention time and degree of matrix effect as analyte in the LC–MS/MS analysis. A series of available resveratrol oligomers such as resveratrol and g-viniferin were investigated; however, they were not used as an IS for various reasons. Finally, carbamazepine was chosen for the method validation and the sample analysis because of the extraction recovery, matrix effect, and retention time under the above conditions.
3.2 LC–MS/MS method validation 3.2.1 Specificity No interfering peaks from six batches of rat plasma or urine were observed at the elution times of vitisin B (1.52 min) and the IS (2.0 min). Figure 2 presents typical chromatograms for drug-free rat plasma/or urine, plasma samples at LLOQ www.jss-journal.com
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Figure 2. Representative chromatograms of vitisin B (I) and carbamazepine (IS) (II) in plasma and urine: (A) double blank plasma or urine, (B) blank plasma or urine spiked with vitisin B at LLOQ (10 ng/mL for plasma and 5 ng/mL for urine), and the IS (50 ng/mL), and (C) a plasma sample from 5 min (vitisin B concentration: 224 ng/mL) or a urine (vitisin B concentration: below LLOQ) sample collected from 0 to 24 h after intraperitoneal injection of 25 mg/kg vitisin B.
(10 ng/mL for plasma and 5 ng/mL for urine), a plasma sample from 5 min (224 ng/mL), and a urine sample collected from 0 to 24 h after intraperitoneal injection of 25 mg/kg vitisin B. The total run time per sample was 3.5 min. 3.2.2 Carryover None of the analytes showed a significant peak (20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. 3.2.3 Linearity of calibration curve, sensitivity, and LLOQ
coefficients (r) of 0.9833–0.9969 in plasma and 0.9977–0.9986 in urine. The representative regression equations for calibration curves during the validation were y = 0.00236x – 0.00519 for plasma and y = 0.00485x – 0.00166 for urine. The backcalculated results for all calibration standards in plasma were
ISSN 1615-9306 · JSSCCJ 38 (11) 1813–2006 (2015) · Vol. 38 · No. 11 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
11 15
www.jss-journal.com
1872 Woong-Kee Choi1 ∗ Kee Dong Yoon1 ∗ Joeng Kee Lee1 Jung Bae Park1 Tae-Hwe Heo1 Choongho Lee2 Soo Kyung Bae1 1 College
of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, Bucheon, South Korea 2 College of Pharmacy, Dongguk University, Goyang, South Korea Received January 19, 2015 Revised March 2, 2015 Accepted March 11, 2015
J. Sep. Sci. 2015, 38, 1872–1880
Research Article
Development and validation of a liquid chromatography with tandem mass spectrometry method for the quantification of vitisin B in rat plasma and urine A new, rapid, and sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of vitisin B and validated in rat plasma and urine using carbamazepine as an internal standard. The plasma (0.05 mL) or urine (0.2 mL) samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Eclipse Plus C18 column (100 × 4.6 mm, 3.5 m) with a mobile phase consisting of acetonitrile and 0.1% formic acid water (60:40, v/v) at a flow rate of 0.7 mL/min. Detection and quantification were performed by mass spectrometry in selected reaction-monitoring mode with positive electrospray ionization. The calibration curves were recovered over the concentration ranges of 10−5000 ng/mL (correlation coefficients, r0.9833) in plasma and 5−2500 ng/mL (r0.9977) in urine, respectively. All validation data, including the specificity, precision, accuracy, recovery, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of vitisin B following intravenous administration of 0.5 and 1 mg/kg and intraperitoneal injection of 5, 10, and 25 mg/kg to rats. This is the first report on the pharmacokinetic properties of vitisin B. The results provide a meaningful basis to evaluate preclinical or clinical applications of vitisin B. Keywords: Liquid chromatography with tandem mass spectrometry / Pharmacokinetics / Plasma / Urine / Vitisin B DOI 10.1002/jssc.201500071
1 Introduction Resveratrol oligomers are characterized by the polymerization of two to eight resveratrol units and are the largest group of oligomeric stilbenes [1, 2]. Resveratrol oligomer polyphenols were mainly isolated from five plant families; namely, Vitaceae, Leguminosae, Gnetaceae, Dipterocarpaceae, and Cyperaceae [1–5]. In addition, resveratrol oligomers were recognized as fungal detoxification products of resveratrol metabolism. These oligomers exhibit diverse biological activities, such as antibacterial, antifungal, anticancer, anti-HIV, Correspondence: Professor Soo Kyung Bae, College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do, 420–743, South Korea E-mail: [email protected] Fax: +82-2-2164-4096
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; CL, total body clearance; Cmax , peak plasma concentration; F, bioavailability; LLOQ, lower limit of quantitation; QC, quality control; RE, relative error; Tmax , time to reach Cmax ; Vdss , steady-state volume of distribution t1/2 , terminal half-life C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and antioxidant effects [6–8]. Thus, it is important to explore the beneficial effects of resveratrol oligomers. Vitisin B, a resveratrol tetramer, was found to have a variety of pharmacological effects including cardiovascular protective [9], antimicrobial [10, 11], anti-inflammatory [12, 13], antileukemic [14], and cholesterol-lowering activities [15], making it a promising candidate for further research. Although many pharmacological data on vitisin B have been published [9–15], no information is available regarding its pharmacokinetic properties in animals. Based on the clinical potential of vitisin B, it is important to understand its pharmacokinetic characteristics. To date, no analytical method for vitisin B in biological fluids has been reported. Several HPLC–UV and LC–MS methods were published to confirm the structures and the detectable amount of vitisin B in plant extracts [13, 14, 16, 17]. However, these methods cannot typically be applied directly to the bioanalysis of vitisin B owing to interference from endogenous materials, insufficient sensitivity, and long retention times. Moreover, these methods have not been validated. Therefore, a more sensitive and rapid analysis method is required for monitoring of vitisin B concentrations in biological samples.
∗ Both
authors equally contributed to this work.
www.jss-journal.com
J. Sep. Sci. 2015, 38, 1872–1880
In this study, a rapid and sensitive LC–MS/MS method to determine vitisin B in rat plasma and urine was developed, validated, and successfully applied to pharmacokinetic studies of vitisin B after intravenous administration (0.5 and 1 mg/kg) and intraperitoneal injection (5, 10, and 25 mg/kg) in rats.
2 Materials and methods 2.1 Materials and reagents Vitisin B (purity: 95.7%) was isolated from Vitis amurensis as described previously [17]. Carbamazepine, dimethylsulfoxide, formic acid, and PEG 400 were purchased from Sigma–Aldrich. Ethyl acetate was obtained from Burdick & Jackson (Morristown, NJ, USA). All solvents were of HPLC grade. All other chemicals were of the highest quality available. Freshly obtained rat plasma (blank plasma) and urine (blank urine) were collected from male Sprague–Dawley rats and stored at –20⬚C before use.
2.2 Calibration standards and QCs Stock solutions of vitisin B and carbamazepine (internal standard, IS) were prepared by dissolving in dimethylsulfoxide at 5 mg/mL, respectively. The IS stock solution was further diluted to 50 ng/mL in acetonitrile for routine use. The working solutions of vitisin B were prepared by serial dilutions of the stock solution with dimethylsulfoxide to obtain the final desired concentrations. Calibration standards and QC samples in plasma were prepared by spiking 2.5 L of appropriate working standard or QC solution with 47.5 L drug-free rat plasma. The ranges of calibration curves in rat plasma were 10, 20, 100, 200, 500, 2000, and 5000 ng/mL. Additionally, the QC samples were prepared in drug-free rat plasma at four different concentration levels of 10 (lower limit of quantification; LLOQ), 30 (low QC), 400 (medium QC), and 4000 ng/mL (high QC) to evaluate the intra- and inter-day precision and accuracy of this method. The calibration standards and QC samples in rat urine were prepared by spiking 10 L of appropriate working standard or QC solution with 190 L of drug-free rat urine. The ranges of calibration curves in rat urine were 5, 10, 50, 100, 250, 1000, and 2500 ng/mL and the QC levels in rat urine were 5 (LLOQ), 15 (low QC), 200 (medium QC), and 2000 ng/mL (high QC). The stock solutions for QC samples were prepared independently from the calibration standards. On the day of analysis, calibration curves for vitisin B in rat plasma or urine were derived from their peak area ratios of vitisin B to the IS versus the respective plasma or urine concentrations of vitisin B using a linear regression with 1/x2 as a weighting factor. The respective QC samples were assayed along with each batch of plasma or urine samples. All prepared plasma or urine samples and stock solutions were stored at –80⬚C (Revco ULT 1490 D-N-S; Western Mednics, Asheville, NC, USA). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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2.3 Sample preparation For plasma samples, 10 L of the IS solution containing 50 ng/mL carbamazepine were mixed with 50 L plasma sample, after which 1 mL of ethyl acetate were added and vortex-mixed for 5 min. After centrifugation (16 000 ×g for 15 min), the organic layer was transferred to another tube and was completely evaporated under a gentle stream of nitrogen gas at 40⬚C. The residue was reconstituted with 100 L of mobile phase and centrifuged (16 000 ×g for 10 min at 4⬚C). Then, a 10 L aliquot was injected into the LC–MS/MS system for analysis. For urine samples, 10 L of the IS (50 ng/mL carbamazepine) were mixed with 200 L urine sample, after which 1.5 mL of ethyl acetate were added and vortex-mixed for 5 min. Other procedures were similar to those used in the analysis of plasma samples.
2.4 LC–MS/MS conditions The samples were analyzed using a API 5500 Q-Trap mass spectrometer (AB SCIEX, Foster City, CA, USA) equipped with a 1260 HPLC system (Agilent Technologies, Wilmington, DE, USA) in ESI mode used to generate positive [M+H]+ . The compounds were separated on a C18 reversed-phase column (Eclipse Plus C18 column, 100 × 4.6 mm i.d., 3.5 m particle size; Agilent) with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (60:40, v/v) at a flow rate of 0.7 mL/min. The column and autosampler temperatures were maintained at 40 and 4⬚C, respectively. The total run time was 3.5 min for each sample. The optimized ion spray voltage and temperature were set at 5500 V and 600⬚C, respectively. The operating conditions, which were optimized by flow injection of vitisin B, were as follows: declustering potential and collision energy were 30 and 30 V for vitisin B and 190 and 25 V for the IS, respectively. The entrance potential and collision cell exit potentials were 10 and 5 V, respectively. Nitrogen gas was used as the nebulizer gas, curtain gas, and collision-activated dissociation gas, which were set at 20, 70, and 30 psi, respectively. The mass transitions used for vitisin B and the IS were m/z 907.3→559.2 and 237.2 → 194.1, respectively. Data acquisition was performed with dwell times of 150 ms, and quadrupoles Q1 and Q3 were set on unit resolution. The analytical data were processed using the Analyst software (version 1.5.2; Applied Biosystems, Foster City, CA, USA).
2.5 LC–MS/MS method validation The validation parameters were selectivity, linearity, sensitivity, accuracy, precision, and matrix effects of the assay and the recovery and stability of vitisin B in rat plasma or urine, in accordance with the US Food and Drug Administration (US FDA) guidance for the validation of bioanalytical methods [18]. www.jss-journal.com
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The specificity was assessed by comparing the chromatograms of six different batches of drug-free rat plasma or urine to ensure that no interfering peaks were present at the retention times of vitisin B(10 ng/mL for plasma and 5 ng/mL for urine) and the IS at the LLOQ. The carryover was investigated by injecting two double blank plasma or urine samples directly after a highest concentration sample from the respective calibration curves (upper limit of quantification; ULOQ) in an analytical run. The carryover test was met if no interfering peaks appeared with areas greater than 20% of the peak areas at the LLOQ level of each analyte and 5% of the IS peak area. Calibration curves were prepared by assaying standard rat plasma or urine samples of vitisin B at the concentration ranges of 10–5000 ng/mL for rat plasma and 5–2500 ng/mL for rat urine. The linearity of each method matched calibration curve was determined by plotting the peak area ratio (y) of vitisin B to the IS versus the nominal concentration (x) of vitisin B. The calibration model was selected based on analysis of data by linear regression with weighting factors (1/x, 1/x2 , and none).The LLOQ of the assay was defined as the lowest concentration on the calibration curve of an analyte with precision (±20%) and accuracy (80–120%) that could be quantitated with an S/N of at least 10. The inter-day precision and accuracy were evaluated by analyzing ten replicates of the LLOQ sample and three different QC samples on five days (two replicates per day).The intra-day precision and accuracy were determined by analyzing six replicates of the LLOQ sample and three different QC samples on the same day. The precision was determined based on the RSD (RSD,%), and the accuracy was expressed as the relative error (RE): RE (%) = (found concentration − theoretical concentration)/theoretical concentration × 100%. The concentrations of QC samples, including LLOQ samples, were determined from the standard calibration curve and were analyzed on the same day. The acceptance criterion was ±15% deviation from the normal value, except at the LLOQ (±20%). To evaluate extraction recoveries and matrix effects, six different batches of drug-free rat plasma or urine were used. Extraction recoveries of vitisin B were determined by comparing the peak areas of extracted QC samples with the peak areas of post-extraction plasma or urine blanks spiked with the corresponding concentrations at three QC levels [19]. The matrix effects on the ionization of the analytes were evaluated by comparing the peak areas of post-extraction blank plasma or urine spiked at the concentrations of the QC samples with the areas obtained by direct injection of the corresponding standard solutions [19]. The matrix effects and extraction recovery of the IS at 50 ng/mL were evaluated using the same method. All assays were performed in sextuplicate. The dilution integrity test was performed to ensure that plasma samples could be diluted with blank plasma without affecting the final concentration. The dilution QC samples (25 000 ng/mL for plasma) were diluted tenfold with blank rat plasma before extraction in six replicates and analyzed. As part of the validation, the replicates had to comply with both C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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precision of 15% and accuracy of 85–115%, similar to other QC samples. Stability tests for stock solutions of vitisin B and the IS were conducted at 4 and –80⬚C. Stability in the stock solution was evaluated by comparing the area response of the analytes (stability samples; stored) with the response of the samples prepared from a fresh stock solution. The solutions were considered stable if the deviation from the nominal value was within ±10.0%. The stabilities of vitisin B in rat plasma or urine were established at low QC and high QC levels using six replicates under the following conditions: (1) short-term storage (8 h at room temperature), (2) long-term storage (90 days at –80⬚C), (3) three freeze–thaw cycles, and (4) post-treatment storage (24 h at 4⬚C). Stability results in plasma were evaluated by measuring the area ratio response (analyte/IS) of the stability samples against freshly prepared QC samples with identical concentrations. Stability data in plasma or urine were acceptable if the deviation from the mean area ratio response of freshly prepared QC samples was within ±15.0%.
2.6 Pharmacokinetic studies in rats Pharmacokinetic studies were performed in rats to verify the applicability of the method. Male Sprague–Dawley rats (eight weeks old, 250–270 g) were purchased from Orient Bio (Sungnam, Gyeonggi-do, South Korea). The protocol for the animal study was approved by the Institutional Animal Care and Use Committee on the Sungsim Campus of The Catholic University of Korea (Bucheon, South Korea). The procedures used for housing and handling were as reported previously [20]. The jugular vein (for drug administration for intravenous study only) and carotid arteries (for blood sampling) of each rat were cannulated with a polyethylene tube (Clay Adams, Franklin Lakes, NJ, USA). Each rat was housed individually in a rat metabolic cage (Daejong Scientific Company, Seoul, South Korea) and allowed to recover from aesthesia for 4–5 h before beginning the experiment. Thus, rats were not restrained in the present study. Vitisin B (dissolved in dimethylsulfoxide: PEG200: normal saline = 2:48:50, v/v) at doses of 0.5 (n = 5) and 1 (n = 5) mg/kg was infused (total infusion volume of 2 mL/kg) over 1 min by the jugular vein to rats. Approximately 0.12 mL of blood from each rat was collected by the carotid artery at 0 (control), 1 (at the end of the infusion), 5, 10, 15, 30, 45, 60, 90, and 120 min after the start of the intravenous infusion of vitisin B. A heparinized 0.9% NaCl-injectable solution (20 units/mL, 0.3 mL) was used to flush the cannula immediately after each blood sampling to prevent blood clotting. The blood samples were immediately centrifuged at 13 000 rpm for 10 min at 4⬚C and the plasma samples (50 L) were stored at –80⬚C until LC–MS/MS analysis. At the end of 24 h, each metabolic cage was rinsed with 10 mL of distilled water and the rinsings were combined with the 24 h urine sample. After measuring the exact volume of the combined urine sample, two 200 L aliquots of the combined urine sample were stored at –80⬚C until LC–MS/MS www.jss-journal.com
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analysis. After the experiments, the rats were euthanized by CO2 gas. Vitisin B (the same solution used in the intravenous study) at doses of 5 (n = 5), 10 (n = 5), and 25 mg/kg (n = 5) was injected intraperitoneally (total injection volume of 3 mL/kg). A blood sample (approximately 0.12 mL) was collected by the carotid artery at 0, 5, 15, 30, 60, 120, 180, 240, 360, 480, 600, 720, 1080, and 1440 min after intraperitoneal injection of vitisin B. Other procedures were as for the intravenous study. Pharmacokinetic parameters including the terminal halflife (t1/2 ), area under concentration–time curve (AUC0–t and AUC0– ), total body clearance (CL), and steady-state volume of distribution (Vdss ) of vitisin B were analyzed using a noncompartmental method using WinNonlin Professional, version 2.1. The maximum plasma concentration (Cmax ) and the time at which the maximum plasma concentration was obtained (Tmax ) were the observed values. The bioavailability (F) for intraperitoneal vitisin B was calculated as: F = AUCip /AUCiv × doseiv /doseip × 100%
(1)
where AUCip is the AUC0– after intraperitoneal injection of vitisin B; and AUCiv is the AUC0– after intravenous administration of vitisin B at a dose of 1 mg/kg. A p value < 0.05 was deemed to be statistically significant using a t-test between the two means for the unpaired data, or a Duncan’s multiple range test of Statistical Package for the Social Sciences (SPSS) posteriori analysis of variance (ANOVA) among the three means for the unpaired data.
3 Results and discussion 3.1 LC–MS/MS optimization To optimize the MS parameters, vitisin B stock solutions were directly injected into the ion source of the mass spectrometer using a syringe pump. In positive-ion mode, vitisin B and the IS gave deprotonated molecular ions, [M+H]+ , as the major species. Final optimization of mass parameters was performed using the automatic tuning tool to obtain maximum sensitivity of selected reaction-monitoring quantification. Figure 1 displays the product ion mass spectra of vitisin B and the IS at their optimal collision energies. To optimize chromatographic conditions, many column types, including C18 , C8 , phenyl-hexyl, cyano, and HILIC columns, and various compositions of mobile phase were evaluated for resolution, peak symmetry, and retention time. The use of an Agilent Eclipse Plus C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% aqueous formic acid (60:40, v/v) resulted in the best peak shape and highest sensitivity. Initially, protein precipitation using methanol and acetonitrile for sample preparation was attempted, but significant lower sensitivity hindered further development. Thus, a LLE procedure was investigated. Several organic solvents, C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Product ion mass spectra of vitisin B (A) and carbamazepine (IS; B) with [M+H]+ at m/z 559.2 and 194.1 as the precursor ions.
including ethyl acetate, ether, dichloromethane, acetone, chloroform, methyl tert-butyl ether, and their mixtures were evaluated. Finally, ethyl acetate was found to be optimal, producing a clean chromatogram for blank plasma or urine samples, constant extraction recovery, and negligible matrix effects. For a natural product with a complex molecular structure, isotope-labeled IS was difficult to obtain. It was challenging to select an appropriate IS with a similar retention time and degree of matrix effect as analyte in the LC–MS/MS analysis. A series of available resveratrol oligomers such as resveratrol and g-viniferin were investigated; however, they were not used as an IS for various reasons. Finally, carbamazepine was chosen for the method validation and the sample analysis because of the extraction recovery, matrix effect, and retention time under the above conditions.
3.2 LC–MS/MS method validation 3.2.1 Specificity No interfering peaks from six batches of rat plasma or urine were observed at the elution times of vitisin B (1.52 min) and the IS (2.0 min). Figure 2 presents typical chromatograms for drug-free rat plasma/or urine, plasma samples at LLOQ www.jss-journal.com
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Figure 2. Representative chromatograms of vitisin B (I) and carbamazepine (IS) (II) in plasma and urine: (A) double blank plasma or urine, (B) blank plasma or urine spiked with vitisin B at LLOQ (10 ng/mL for plasma and 5 ng/mL for urine), and the IS (50 ng/mL), and (C) a plasma sample from 5 min (vitisin B concentration: 224 ng/mL) or a urine (vitisin B concentration: below LLOQ) sample collected from 0 to 24 h after intraperitoneal injection of 25 mg/kg vitisin B.
(10 ng/mL for plasma and 5 ng/mL for urine), a plasma sample from 5 min (224 ng/mL), and a urine sample collected from 0 to 24 h after intraperitoneal injection of 25 mg/kg vitisin B. The total run time per sample was 3.5 min. 3.2.2 Carryover None of the analytes showed a significant peak (20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. 3.2.3 Linearity of calibration curve, sensitivity, and LLOQ
coefficients (r) of 0.9833–0.9969 in plasma and 0.9977–0.9986 in urine. The representative regression equations for calibration curves during the validation were y = 0.00236x – 0.00519 for plasma and y = 0.00485x – 0.00166 for urine. The backcalculated results for all calibration standards in plasma were
