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Rapid quantification of resveratrol in mouse plasma by ultra high pressure liquid chromatography (UPLC) coupled to tandem mass spectrometry.

Objective: The objective of this study was to develop a rapid and sensitive method for the quantification of resveratrol, a polyphenolic compound with multiple health beneficial effects, in mouse plasma.

Methods: We used reversed-phase ultra high pressure liquid chromatography with tandem mass spectrometry detection for the determination of resveratrol levels in mouse plasma. An Agilent Zorbax Eclipse Plus [C.sub.18] column (2.1 mm x 50 mm, 1.8 [micro]m) was used as the stationary phase. The mobile phase consisted of a gradient formed using 1 mM ammonium fluoride and methanol.

Results: Using this improved method, we obtained a retention time of 2.2 min and a total run time of 5 min, for resveratrol. The calibration curve for resveratrol showed a linear range from 0.5 to 100 ng/mL. The average coefficient of variation was 6% for interday variation and 4% for intraday variation. The recovery for resveratrol in mouse plasma was 85 [+ or -] 10% (mean [+ or -] standard deviation).

Conclusion: The method presented herein allows a rapid and very sensitive quantification of resveratrol in mouse plasma at concentrations as low as 500 ppt.

Key words: Resveratrol, Quantification, UPLC, Plasma, Mass spectrometry

Objetivo: El objetivo del presente estudio consistia en desarrollar un metodo rapido y sensitivo para la cuantificacion de resveratrol, un compuesto de tipo polifenolico con multiples propiedades beneficiosas para la salud, en plasma de raton. Metodos: Utilizamos cromatografia liquida con presion ultra alta acoplada a espectrometria de masa en serie, para la determination de los niveles de resveratrol en plasma de raton. Una columna Agilent Zorbax Eclipse Plus [C.sub.18] (2.1 mm x 50 mm, 1.8 [micro]m) fue utilizada como fase estacionaria. La fase movil consistio de un gradiente de fluoruro de amonio 1 mM y metanol. Resultados: Obtuvimos un tiempo de retention de 2.2 min para resveratrol y el tiempo total de corrida fue de 5 min. La curva de calibration para resveratrol se comporto de manera lineal en un rango de 0.5 a 100 ng/mL. El coeficiente de variation promedio fue 6% para la variation entre dias y de 4% para la variation en un mismo dia. El porciento de recuperation de resveratrol en plasma de raton fue de 85 [+ or -] 10% (promedio [+ or -] desviacion estandar). Conclusion: El metodo que hemos desarrollado permite la cuantificacion de resveratrol en plasma de raton de manera rapida y sensitiva. Utilizando este metodo se pueden detectar cantidades de resveratrol tan bajas como 500 ppt.

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Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a polyphenol particularly enriched in red wine, and also found in berries, grapes, and peanuts. It has been widely studied during the past years due to its antioxidant, anti-aging, anti-inflammatory, cardioprotective, neuroprotective, antidiabetic, and anticancer properties (1). Among resveratrol's health benefits, its cardioprotective and anticancer qualities have received special attention. Resveratrol acts as a cardioprotective agent by a plethora of activities that prevent atherosclerosis and coronary heart disease. Resveratrol: 1) inhibits low density lipoprotein oxidation, a primary event in the initiation of atherosclerosis; 2) inhibits platelet aggregation, allowing for the rapid repair of injuries in the vascular endothelium; 3) suppresses proliferation of smooth muscle cells and pulmonary aortic endothelial cells, which is necessary for atherogenesis; and 4) induces nitric oxide synthase (2).

The anticancer properties of resveratrol have been demonstrated both in vitro and in vivo by many research groups. Resveratrol inhibits all stages of tumorigenesis by modulating cell division and growth, apoptosis, angiogenesis and metastasis (3,4). Additionally, resveratrol has proven to be effective as a chemosensitization agent against several types of cancer. Resveratrol's ability to chemosensitize cancers to therapy has been attributed to the regulation of many signaling molecules including drug transporters, cell survival and cell proliferation regulators, and members of the nuclear factor kappa B and signal transducer and activator of transcription 3 signaling pathways (5).

Resveratrol has the capability to act in an estrogenic or antiestrogenic manner to inhibit or promote breast cancer progression, dependent on concentration, and can bind to and regulate gene transcription through estrogen receptor [alpha] and [beta] isoforms (6-8). Therefore, resveratrol is of particular interest for gynecological cancers, such as breast cancer. Our laboratory has demonstrated that resveratrol, similar to estrogen, regulates actin structures and focal adhesions that are relevant for breast cancer cell migration and invasion. We reported an inhibitory role for high concentrations of resveratrol and a promotional role for low concentrations of resveratrol in signaling to the actin cytoskeleton and breast cancer cell migration (9-11). Moreover, we recently reported that resveratrol increases tumor growth and metastases from mammary fat pad tumors, established with human breast cancer cells, in nude mice (12). Therefore, it is critical to identify the effective concentrations of resveratrol in the circulation following consumption of resveratrol-rich foods or dietary supplements.

Resveratrol's bioavailability is relatively low; however, due to its therapeutic potential, many efforts are currently directed towards the generation of novel delivery systems for resveratrol (13,14). Since resveratrol's dual estrogenic/antiestrogenic role appears to be concentration dependent, it is important to delineate its anticancer effects at abroad range of concentrations. Correlation of the physiological effects of resveratrol in organisms with circulating plasma levels may be used as a prognostic tool for resveratrol effects in vivo. However, thus far detection and quantification of low levels of resveratrol has been limited by the sensitivity of the available methods. Sensitivity of detection of resveratrol in plasma and urine becomes extremely important, especially for the study of cancer promotion by low levels of resveratrol. To address this need, we developed a rapid ultra high pressure liquid chromatography (UPLC)-tandem mass spectrometry (MS) method that requires minimal sample preparation and allows the quantification of resveratrol in mouse plasma at lower levels and/or shorter retention times than those previously reported by similar methodologies (15-19).

Materials and Methods

Chemicals and reagents

Resveratrol (Figure 1) was purchased from LKT Laboratories, St. Paul, MN. Deuterated resveratrol ([resveratrol-d.sub.4]) (Fig. 1) from Cayman Chemical, Ann Arbor, MI, was used as internal standard (IS). Liquid chromatography (LC)-MS CHROMASOLV[R] grade [greater than or equal to] 99.9% methanol and acetonitrile (ACN) and [greater than or equal to] 99.99% ammonium fluoride were purchased from Sigma-Aldrich, St Louis, MO. Mouse plasma containing sodium citrate was obtained from Equitech-Bio, Inc, Kerrville, TX.

Instrument

The UPLC MS/MS system consisted of an Agilent 1290 dual pump chromatograph with a 6460 Triple Quad LC/MS (Agilent Technologies, Santa Clara, CA). Agilent MassHunter Workstation software was used to control both the instrument and data acquisition. Qualitative and quantitative data analysis was performed with Agilent Mass Hunter Workstation Software, Version B.04.00/Build 4.0.225.0 for QQQ.

Standard solutions

Stock solutions of resveratrol were prepared at concentrations of 1 mg/mL, 100 [micro]g/mL, and 1 [micro]g/mL. The IS stock solution was prepared at 4 [micro]g/mL. All stock solutions were prepared in 50% methanol and stored at -20[degrees]C in the dark. Solutions of resveratrol in mouse plasma were prepared by diluting the stock solutions prepared in 50% methanol. Plasma solutions were prepared at concentrations of 0.5, 1, 5, 10, 50, and 100 ng/mL resveratrol for the calibration curve; and at concentrations of 7, 25, and 70 ng/mL resveratrol for calibration control. Once prepared, plasma solutions were dispensed into 100 [micro]L aliquots and stored at -80[degrees]C.

Sample preparation

A total of 2.5 [micro]L of IS (4 pg/mL stock solution) were added to 100 [micro]L of plasma to obtain a final IS concentration of 100 ng/mL. Then, 400 [micro]L of ACN were added and the sample was vortex-mixed for 5 s and spun at 9,000 x g (4[degrees]C) for 10 min to precipitate proteins. Following protein precipitation, samples were centrifuged for 10 minutes at 3,300 rpm at 4[degrees]C, the supernatant was carefully transferred to a glass tube and the solvent was dried at room temperature using a vacuum concentrator. Once dry, samples were reconstituted in 100 [micro]L of 50% methanol, filtered through a 0.45 pm nylon filter, and transferred to appropriate vials for injection into the UPLC MS/MS system.

Ultra high pressure liquid chromatography

Chromatographic separation was achieved by injecting 5 [micro]L of sample into a Zorbax Eclipse Plus [C.sub.18] column (2.1x50 mm, 1.8 [micro]m, Agilent Technologies, Santa Clara, CA). The column temperature was set to 40[degrees]C. The mobile phase consisted of a gradient of 1 mM ammonium fluoride in distilled water (solvent A) and 100% methanol (solvent B) at a flow of 0.5 mL/min. The gradient elution was carried out as follows: 0 min, 0% B; 2 min, 30% B; 2.5 min, 70% B; 2.6 min, 95% B; 3.5 min, 95% B; and finally 3.6 min, 30% B for re-equilibrating. Retention time for both resveratrol the reseveratrol-[d.sub.4] was 2.22 and 2.17 min, respectively.

Mass spectrometry

An Agilent 6460 employing electrospray ionization (ESI) with Jet Stream technology was used in the negative ionization mode. The instrument settings were as follows: time filtering width 0.07 s; gas temperature 350[degrees]C; gas flow 10 L/min, nebulizer gas 20 psi; sheath gas heater 400[degrees]C; sheath gas flow 12 L/min; capillary voltage 4000 V; cell accelerator voltage (CAV) 7 V; dwell time 100 ms; and collision energy (CE) 12 V for resveratrol quantifier (227 [right arrow] 185), 20 V for resveratrol qualifier (227 [right arrow] 143), and 24 V for the IS (23 [right arrow] 147).

Results and Discussion

Resveratrol analysis and detection

Resveratrol and resveratrol-[d.sub.4] were eluted at 2.22 min and 2.17 min respectively with 70% percent of organic solvent B through a [C.sub.18] column set at 40[degrees]C (Figure 2). For quantification, negative ESI was used with ammonium fluoride at 1 mM as the aqueous solvent, which has been previously reported to substantially improve ionization in the negative ESI mode (20). Only resveratrol and IS precursor ions with mass to charge (m/z) of 227 and 231, respectively, and at unit resolution were allowed from MSI to MS2. Product ions 185, 143, and 147 m/z with unit resolution were detected and later used as resveratrol quantifier peak, resveratrol qualifier peak, and IS peak, respectively. The run time was only 5 min for each sample, representing a great improvement over previously described methodologies for quantification of resveratrol in plasma (16,17).

Linearity

Six calibration runs for resveratrol in mouse plasma were performed using standards of 0.5, 1,5, 10, 50, and 100 ng/mL resveratrol, containing IS at 100 ng/mL. All calibration curves were linear over the measured range, and are defined by the following equationy = (0.018289 [+ or -] 0.000683)x+ (-0.0009212 [+ or -] 0.0016098) (mean [+ or -] standard deviation (SD) for slope and y intercept); and presented a correlation coefficient ([r.sup.2]) of 0.99916 [+ or -] 0.00055 (mean + SD) (Figure 3).

Limit of detection and quantification, precision and accuracy

The limit of detection (LOD) and limit of quantification (LOQ) for resveratrol in plasma was 0.25 and 0.5 ng/mL, respectively. Interday and intraday precision and accuracy showed a coefficient of variation (CV) of 6 and 4% (in average) and a relative error (RE) of 1.3 and 1.4% (in average), respectively (Table 1).

Recovery

To calculate the percentage of recovery for resveratrol in mouse plasma, we used the ratio of the concentration calculated for the extracted standards versus the concentration calculated for the neat standards (5, 50, and 100 ng/ mL in 50% methanol). To prepare the extracted standards, we used mouse plasma that underwent the sample preparation process but was reconstituted in resveratrol standards of 5, 50, and 100 ng/ mL in 50% methanol (neat standards). Five replicates of each standard were prepared and each one was injected twice; all standards were assayed in the same analytical run. The percent of recovery for resveratrol in mouse plasma was high, as shown by the reported values of 82, 76, and 96% recovery for 5, 50, and 100 ng/mL, respectively; average percent of recovery was 85 [+ or -] 10% (Table l).

Stability

Several tests were performed to evaluate the stability of resveratrol: benchtop and freeze/thaw stability in plasma and post-preparative reinjection reproducibility in reconstitution solvent were determined. For the benchtop stability test, 1 and 100 ng/mL resveratrol standards (5 replicates; each injected once) were thawed and, after 6 h at room temperature, subjected to the sample preparation protocol (see methods). Our results show a CV of 14 and 4% and a RE of 6 and 2% for the 1 and 100 ng/mL resveratrol standards, respectively (Table 1). On the other hand, the freeze/thaw stability test used 25 and 100 ng/mL resveratrol standards (one replicate; each injected 5 times) that underwent 5 freeze/thaw cycles (15 min at -80[degrees]C/15 min at room temperature), prior to sample preparation. The CV was 3 and 2% and the RE was 11 and 1% for 25 and 100 ng/mL resveratrol standards, respectively (Table 1). Finally, the calculated concentration of 25 and 70 ng/mL resveratrol standards (two replicates; each injected twice) at 0 h and after 96 h at room temperature were compared for the determination of post-preparative reinjection reproducibility of resveratrol in reconstitution solvent. After 96 h, the percent of recovery was 99 [+ or -] 7% (mean [+ or -] SD) and 98 [+ or -] 6% for 25 and 70 ng/mL resveratrol standards, respectively (Table 1). These results suggest that resveratrol is stable after 6 h in plasma and after 96 h in reconstitution solvent at room temperature (Table 6). Additionally, resveratrol was proven to be stable after 5 freeze/thaw cycles. The robust stability of resveratrol greatly simplifies sample handling during preparation and allows for the preparation of multiple samples simultaneously.

Application of the method

The described method allows for the rapid quantification of low levels of resveratrol in mouse plasma. The reported methodology allows the quantification of resveratrol at lower levels and/or shorter retention times than previously reported methodologies (15-19).Our method has a total run time of only 5 min with a LOQ of 0.5 ng/mL or 2.19 nM. In comparison, other methods report longer run times and higher LOQs. For instance, the method published by Juan ME, et al. (21) to detect resveratrol in rat plasma by high pressure liquid chromatography (HPLC) has a run time of >20 min and a LOQ of 5.77 nM, the method reported by Singh G, et al. (22) to detect resveratrol in human plasma by HPLC has a run time of 10 min and a LOQ. of 8 ng/mL, and the method reported by Menet MC, et al. (23) to detect resveratrol in mouse plasma by UPLC has a run time of <10 min and a LOQ of 25 nM. Our rapid and sensitive methodology is expected to enable a more accurate correlation between the plasma levels of resveratrol and its anti-cancer effects. This in turn, will provide a better understanding of the concentration-dependency of resveratrol's health beneficial properties. This method will be used in our studies to determine resveratrol's circulating plasma levels in mammary tumor-bearing immonucompromised mice orally treated with low and moderate concentrations of resveratrol.

Conclusion

Herein we have reported a reliable, rapid and sensitive method for the quantification of resveratrol in mouse plasma using an UPLC MS/MS system. The high recovery, speed, and sensitivity of the method combined with the reported stability of resveratrol are characteristics that render this method a practical tool for the identification and quantification of resveratrol. The described methodology can be further developed to quantify resveratrol in human plasma for pharmacokinetic studies and large scale clinical trials.

Acknowledgments

This study was funded by the Department of Defense/Breast Cancer Research Program awards W81XWH-08-01-0258 to LCP and W81XWH-07-1-0330 to SD and by NIH NCRR G12-RR03051 to UPRMSC.

References

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(4.) Kundu JK, Surh YJ. Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives. Cancer Lett. 2008;269:243-261.

(5.) Gupta SC, Kannappan R, Reuter S, et al. Chemosensitization of tumors by resveratrol. Ann N Y Acad Sci 2011; 1215:150-160.

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(7.) Bowers JL, Tyulmenkov W, Jernigan SC, et al. Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta. Endocrinology 2000;141:3657-3667.

(8.) Levenson AS, Gehm BD, Pearce ST, et al. Resveratrol acts as an estrogen receptor (ER) agonist in breast cancer cells stably transfected with ER alpha. Int J Cancer 2003;104:587-596.

(9.) Azios NG, Krishnamoorthy L, Harris M, et al. Estrogen and resveratrol regulate Rac and Cdc42 signaling to the actin cytoskeleton of metastatic breast cancer cells. Neoplasia 2007;9:147-158.

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(12.) Castillo-Pichardo L, Cubano LA, Dharmawardhane S. Dietary grape polyphenol resveratrol increases mammary tumor growth and metastasis in immunocompromised mice. BMC Complement Altern Med 2013;13:6-15.

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(16.) Boocock DJ, Patel KR, Faust GE, et al. Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 2007;848:182-187.

(17.) Lin HS, Ho PC. A rapid HPLC method for the quantification of 3,5,4-trimethoxy-trans-stilbene (TMS) in rat plasma and its application in pharmacokinetic study. J Pharm Biomed Anal 2009;49:387-392.

(18.) Chen X, He H, Wang G, et al. Stereospecific determination of cis- and trans-resveratrol in rat plasma by HPLC: application to pharmacokinetic studies. Biomed Chromatogr 2007;21:257-265.

(19.) Huang H, Zhang J, Chen G, et al. High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract. Biomed Chromatogr 2008;22:421-427.

(20.) Yanes O, Tautenhahn R, Patti GJ, et al. Expanding coverage of the metabolome for global metabolite profiling. Anal Chem. 2011;83:2152-2161.

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(22.) Singh G, Pai RS, Pandit V. Development and validation of a HPLC method for the determination of trans-resveratrol in spiked human plasma. J Adv Pharm Technol Res 2012;3:130-135.

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Linette Castillo-Pichardo, PhD * ([dagger]); Suranganie Dharmawardhane, PhD *; Jose F. Rodriguez-Orengo, PhD * ([double dagger])

* Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico; ([dagger]) Department of Pathology and Laboratory Medicine, Universidad Central del Caribe, Bayamon, Puerto Rico; ([double dagger]) Fundacion de Investigation de Diego, 998 Munoz Rivera Avenue, San Juan, Puerto Rico

The authors have no conflict of interest to disclose.

Address correspondence to: Linette Castillo-Pichardo, PhD, Assistant Professor, Department of Pathology and Laboratory Medicine, Universidad Central del Caribe, PO Box 60327, Bayamon, PR 00960. Email: linnettecastillo@yahoo.com

Table 1. Method validation parameters for resveratrol in mouse plasma

                                  Resveratrol (ng/mL)

                       0.50   1      5      10     25

                        Mean value for calculated concentration
Validation parameter                 or % recovery

Interday precision
and accuracy           0.51   1.01   4.95   9.67
  SD                   0.06   0.13   0.24   0.49
  %CV                  11     12     5      5
  %RE                  2      1      1      3

Intraday precision
and accuracy                  1.01   5.15   9.67
  SD                          0.11   0.09   0.37
  %CV                         11     2      4
  %RE                         1      3      3

Recovery (%)                         82
Benchtop stability            0.94
  SD                          0.13
  %CV                         14
  %RE                         6

Freeze/thaw
stability                                          27.70
  SD                                               0.78
  %CV                                              3
  %RE                                              11

Post-preparative
reinjection
reproducibility (%
recovery after 96h)                                99 [+ or -] 7

                                     Resveratrol (ng/mL)

                       50      70              100      Average

                           Mean value for calculated concentration
Validation parameter                    or % recovery

Interday precision
and accuracy           49.74                   100.50
  SD                   1.16                    1.89     0.66
  %CV                  2                       2        6
  %RE                  1                       0        1.3

Intraday precision
and accuracy           49.89                   100.28
  SD                   0.96                    2.24     0.75
  %CV                  2                       2        4
  %RE                  0                       0        1.4

Recovery (%)           76                      96       85 [+ or -] 10
Benchtop stability                             102.33
  SD                                           4.45     2.29
  %CV                                          4        9
  %RE                                          2        4

Freeze/thaw
stability                                      98.89
  SD                                           1.58     1.18
  %CV                                          2        2.5
  %RE                                          1        6

Post-preparative
reinjection
reproducibility (%
recovery after 96h)            98 [+ or -] 6
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Author:Castillo-Pichardo, Linette; Dharmawardhane, Suranganie; Rodriguez-Orengo, Jose F.
Publication:Puerto Rico Health Sciences Journal
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Date:Dec 1, 2014
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