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. 2026 Mar 9;11(11):18024–18035. doi: 10.1021/acsomega.5c12760

Validated RP-HPLC-UV Method for the Quantification of Trans-Resveratrol in Plasma: Application to Comprehensive Stability and Comparative Pharmacokinetic Studies

Adilah Marwa †, Mahdi Jufri †, Witta Kartika Restu ‡, Najihah binti Mohd Hashim §, Anton Bahtiar †,*
PMCID: PMC13019402  PMID: 41908373

Abstract

Trans-resveratrol’s (trans-RES) therapeutic potential is limited by poor bioavailability and photolability. We developed and validated a robust RP-HPLC method, in accordance with the ICH M10 guideline, for the quantification of trans-RES in plasma. Excellent chromatographic separation of the trans- and cis-isomers was achieved using a liquid–liquid extraction procedure, followed by isocratic elution with a mobile phase consisting of 0.05% formic acid in water–acetonitrile (40:60, v/v). The method demonstrated high linearity (R 2 > 0.999) over the concentration range of 0.025 to 50 μg/mL. The method also demonstrated precision (%CV < 5%), accuracy (93.90–98.19%), and sensitivity (LLOQ 0.025 μg/mL). Stability studies confirmed trans-RES’s susceptibility to ultraviolet (UV)-induced isomerization, necessitating the implementation of photostability controls. The method’s environmental impact was evaluated using the Analytical GREEnness (AGREE) metric, yielding a favorable score of 0.7 that highlights its alignment with green chemistry principles. This fully validated protocol was successfully applied in the first comprehensive pharmacokinetic study comparing five administration routes in rats. Intraperitoneal administration yielded the highest bioavailability (84.92 ± 4.87%), significantly exceeding that of the oral route (4.91 ± 0.22%). Flip-flop kinetics observed for all nonintravenous routes indicated absorption-limited disposition. The validated method thus provides a reliable, sustainable bioanalytical tool, while the pharmacokinetic insights underscore the imperative for strategic formulation development to overcome bioavailability and stability limitations, thereby advancing the translational potential of trans-RES.

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Introduction

Trans-resveratrol (trans-RES), a polyphenolic compound of the stilbene class with the chemical formula C14H12O3, is a phytoalexin that was first isolated from the roots of Veratrum grandiflorum in 1940. , This compound is found in more than 70 plant species, with the highest concentrations found in grapes (3.66 × 10–2 mg/g), berries (4.209 × 10–2 mg/g), peanuts (1.5 × 10–2 mg/g), and melinjo (3.269 × 10–2 mg/g). Research interest in this compound increased significantly after a 1992 publication that revealed its cardioprotective activity in red wine. Its structural similarity to estrogen allows interaction with estrogen receptors, underpinning its various pharmacological activities, including anti-inflammatory, anticancer, neuroprotective, and antidiabetic. , However, despite this promising therapeutic potential, the clinical application of trans-RES is severely limited by its unfavorable pharmacokinetic profile, particularly its low oral bioavailability.

Specifically, the extremely low oral bioavailability of trans-RES (<1%) is primarily due to its limited water solubility and extensive presystemic metabolism in the liver, where it undergoes rapid conjugation into pharmacologically inactive sulfate and glucuronide metabolites. , For example, Peñalva et al.’s studies show that after oral administration of 15 mg/kg body weight of resveratrol to rats, only a small amount (<5 ng/mL) remains inactive form in the blood. This rapid metabolism causes most of the resveratrol to circulate as inactive conjugates. Similar findings were reported in another study in a rat model with suboptimal pharmacokinetic parameters, showing T max 0.25 h, C max 147.33 ± 25.81 ng/mL, and T 1/2 1.17 ± 0.90 h at a dose of 50 mg/kg–1 per Oral. Importantly, few systematic studies compare pharmacokinetics across administration routes, hindering a complete understanding of the disposition and formulation design.

In addition to these pharmacokinetic challenges, advances in mapping the pharmacokinetic profile of trans-RES are limited by the available analytical methods. While the LC-MS/MS method employed by Chung et al. and Dadge et al. demonstrates high sensitivity, it necessitates costly instrumentation and specialized technical expertise. , Due to the accessibility and cost constraints of the LC-MS/MS method, researchers have developed more practical alternatives using high-performance liquid chromatography (HPLC). Jagwani et al. developed an HPLC method to quantify trans-RES in human and rat plasma. However, this method could not measure trans-RES concentrations below 50 ng/mL in biological matrices with adequate linearity. Meanwhile, the HPLC method developed by Gadag et al. achieved high sensitivity (LOQ: 5 ng/mL); however, gradient elution increased instrumental complexity and prolonged analysis time (15 min per sample). Another important factor often overlooked is the isomeric stability of trans-RES. When exposed to UV light, this compound readily undergoes isomerization to cis-resveratrol (cis-RES). , Since these two isomers have different pharmacokinetic profiles and biological activities, failing to control and monitor isomerization during analytical and pharmacokinetic studies can lead to inaccurate, nonreproducible results. Unfortunately, most existing studies have not adequately addressed this aspect of isomeric stability in their analytical methods.

Therefore, to address the existing research gap, we will develop a comprehensive validation method for quantifying trans-RES in biological matrices using HPLC. This method will be comprehensively validated in accordance with the International Council for Harmonization (ICH) M10 guidance to ensure adequate validation parameters, including specificity, linearity, accuracy, precision, and the lower limit of quantification (LLOQ), especially at low concentrations. Furthermore, the environmental impact of the method will be evaluated using the Analytical GREEnness (AGREE) metric to ensure its alignment with Green Analytical Chemistry principles. In addition, this study will characterize the pharmacokinetics of trans-RES across various routes of administration (oral, intravenous, intraperitoneal, subcutaneous, and intramuscular) to provide a comprehensive understanding of its pharmacokinetic profile. To the best of our knowledge, no studies have reported the stability of trans-RES in plasma against UV exposure. Therefore, this study will evaluate photolytic stability to assess the susceptibility of samples to photolytic degradation and to ensure sample integrity during preparation and analysis. Thus, this study is expected not only to overcome the limitations of existing analytical methods but also to provide comprehensive pharmacokinetic and stability data in accordance with international standards, thereby supporting the development of more effective and stable trans-RES formulations.

Materials and Methods

Materials

Resveratrol (≥99% purity) was obtained from Sigma-Aldrich (St. Louis, MO), while trans-RES (99.5% purity) was obtained from Xi’an Natural Field Bio-Tech, China. HPLC-grade organic solvents, such as acetonitrile (ACN), formic acid 98%, and ethyl acetate, were procured from Merck Ltd., India. Sodium hydroxide was purchased from Spectrochem, India. All other chemicals and reagents were analytical grade and used without further purification.

Methods

Optimization of Extraction Methods

Human plasma was obtained by centrifuging whole blood at 3500 rpm for 10 min. Resveratrol was added to the plasma to achieve a final concentration of 50 μg/mL. For the protein precipitation (PPT) method, 200 μL of added plasma was mixed with 20 μL of the carbamazepine internal standard (IS). Acetonitrile-to-plasma ratios were evaluated (1:1, 1:5, 1:10, and 1:15 v/v). The mixture was vortexed for 10 min and centrifuged at 10,000 rpm for 10 min. HPLC directly analyzed the resulting supernatant. In the liquid–liquid extraction (LLE) method, 200 μL of plasma supplemented with IS was mixed with 20 μL of IS and 100 μL of 0.5 N NaOH. Different ratios of ethyl acetate/plasma were tested (1:1, 1:5, 1:10, and 1:15 v/v). The mixture was vortexed for 10 min and centrifuged at 10,000 rpm for 10 min. The organic phase was collected and evaporated to dryness under a stream of nitrogen gas at 50 °C. The residue was redissolved in 500 μL of mobile phase before HPLC analysis.

Chromatographic Condition

The maximum absorption wavelength (λmax) of trans-RES was determined to be 306 nm through spectral scanning from 280 to 400 nm using a UV–vis spectrophotometer (Shimadzu UV-1800), corresponding to the strongest π→π* electronic transition in its conjugated aromatic structure. This wavelength was selected for quantification to ensure maximum sensitivity and linearity according to the Beer–Lambert principle. ,

Chromatographic separation was performed using an Agilent HPLC system equipped with a ZORBAX Eclipse Plus RP-C18 column (250 × 4.6 mm2, 5 μm) maintained at 30 °C. A gradient elution program was implemented with a mobile phase consisting of water and acetonitrile in varying ratios (50:50, 60:40, and 70:30 V/V) at a flow rate of 1.0 mL/min for 11 min. System suitability tests confirmed optimal chromatographic performance with respect to retention time, peak symmetry, resolution, and theoretical plates, particularly for the separation of trans- and cis-RES isomers.

Preparation of Calibration Standards and Quality Control Samples

Calibration standards and quality control (QC) samples were prepared by spiking drug-free human and rat plasma with appropriate volumes of resveratrol working standard solutions to achieve final concentrations of 0.025, 0.05, 0.5, 1, 2, 5, 10, 25, and 50 μg/mL for the calibration curve along with QC samples at low (LQC, 0.075 μg/mL), medium (MQC, 25 μg/mL), and high (HQC, 37.5 μg/mL) concentrations. The internal standard (IS) concentration was maintained at 20 μg/mL in all samples. Extraction was performed using a standardized protocol: 200 μL of plasma was mixed with 20 μL of IS and 100 μL of 0.5 N NaOH. The mixture was extracted with 2,200 μL of ethyl acetate, vortexed for 10 min, and centrifuged at 10,000 rpm for 10 min. The organic supernatant was evaporated to dryness under nitrogen gas at 50 °C, and the residue was reconstituted in 500 μL of mobile phase before HPLC analysis.

Animals

Sprague–Dawley female rats (170–200 g) obtained from Dramaga Agri Satwa were used in this study. Rats were acclimatized at the animal facility of the Faculty of Pharmacy, University of Indonesia, under standard temperature conditions (25 ± 2 °C), fed and watered ad libitum for 2 weeks. At the end of each experiment, the animals were sacrificed with a lethal dose of ketamine. The Health Research Ethics Committee of the Faculty of Medicine, University of Indonesia, approved the research protocol.

Bioanalytical Validation

The bioanalytical method for trans-RES quantification was validated in accordance with ICH M10. Full validation was performed for human plasma to evaluate specificity, linearity, accuracy, precision, and stability. Specificity was confirmed by analyzing blank plasma samples, which showed no interference at trans-RES and internal standard retention times. Linearity was established using a nine-point calibration curve. Recovery studies assessed accuracy by comparing the analytical responses of extracted quality control samples with those of unextracted standard solutions at equivalent concentrations. Precision was evaluated through intraday and interday analyses with six replicates at each QC concentration level. Comprehensive stability studies included short-term (24 h at 25 °C), long-term (14 days at −20 °C), freeze–thaw (three cycles between −20 and 25 °C), and autosampler stability (24 h at 25 °C).

Partial validation was conducted for rat plasma, focusing on key parameters including specificity, linearity, accuracy, precision, lower limit of quantification (LLOQ), and a system suitability test. This partial validation approach was implemented based on the previous full validation in human plasma and the acceptable matrix similarities between human and rat plasma, as per the regulatory guideline.

Measurement of Trans-RES Stability under UV Exposure

trans-RES powder in plasma was exposed for 0, 5, 15, 30, 45, and 60 min to 365 nm UVA light in a Benchtop 2UV Transilluminator (LMS-20, UVP Ltd., AnalytikJena, Jena, Germany). Resveratrol chromatograms were analyzed by using a validated HPLC method.

Application of the Pharmacokinetic Method Study in Rats

The developed method was used to analyze trans-RES in plasma. Pharmacokinetic and tissue distribution studies were conducted after administration of trans-RES at a dose of 40 mg/kg BW in Sprague–Dawley rats (n = 3) via oral, subcutaneous (SC), intramuscular (IM), intravenous (IV), and intraperitoneal (IP) routes. For the pharmacokinetic study, at predetermined time intervals (0, 0.08, 0.17, 0.33, 0.5, 2, 4, 8, 12, and 24 h), blood samples (∼0.5 mL) were taken from the retroorbital plexus and collected in EDTA-containing tubes. The plasma was then separated by centrifugation. The amount of the drug was quantified using a validated HPLC method.

Greenness Assessment of the Developed Method

The environmental impact of the developed RP-HPLC-UV method was evaluated using the Analytical Greenness (AGREE) metric (version 0.5), a tool based on the 12 principles of green analytical chemistry. The assessment encompassed critical parameters, including sample preparation, specifically solvent type, volume, and energy use, along with chromatographic conditions such as mobile phase composition, flow rate, and column dimensions. It also accounted for waste generation, reagent safety, and overall energy consumption during the analysis. Scores were calculated using the AGREE calculator, with results approaching 1 indicating a more environmentally sustainable procedure.

Statistical Analysis

For parametric data involving two groups, a t-test was used for independent or paired samples. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s posthoc test for multiple comparisons. For nonparametric data, the Mann–Whitney U test was used for independent groups, and the Wilcoxon signed-rank test was used for paired samples. All statistical analyses were performed using SPSS software, version 26. The statistical significance level was set at p < 0.05.

Result and Discussion

Optimization Extraction Methods

This study demonstrates the superior performance of liquid–liquid extraction (LLE) over protein precipitation (PPT) for sample preparation and subsequent chromatographic analysis of trans-RES in human plasma. The systematic evaluation of plasma-to-solvent ratios revealed that LLE achieved optimal and consistent extraction recovery at a ratio of 1:10 (95.2% ± 2.1%), significantly outperforming PPT, which reached only 75.3% ± 3.4% at its optimal 1:10 ratio (Figure ). This enhanced efficiency is explained by the Nernst Partition Law, whereby the analyte preferentially distributes into the organic phase based on its partition coefficients, enabling more complete extraction from the complex plasma matrix. In contrast, the PPT method, while operationally simpler, is inherently limited by the phenomenon of coprecipitation, where analytes are entrapped within the protein pellet, leading to inconsistent and generally lower recovery yields.

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Comparison of the trans-RES extraction recovery: protein precipitation (PPT) and liquid–liquid extraction (LLE) across different solvent ratios.

The cleaner extract obtained through LLE directly translates to superior chromatographic outcomes, as quantified in Table . Specifically, LLE achieved a significantly higher resolution (R s = 7.85 ± 0.20) than PPT (R s = 6.10 ± 0.045), indicating an exceptional separation efficiency between the closely eluting geometric isomers, which is critical for accurate quantification. − This enhancement in resolution is corroborated by a higher number of theoretical plates (NTP = 13,650 ± 127.89 for LLE and 11,843 ± 100.80 for PPT), reflecting reduced peak broadening and superior column efficiency, consistent with chromatographic theory, which posits that cleaner samples minimize on-column interactions that contribute to band broadening. , Both methods yielded excellent and comparable peak symmetry, as indicated by tailing factors near unity (0.93–1.00), ensuring reliable integration. The collective data spanning recovery, resolution, and efficiency robustly indicate that LLE provides a more reliable and effective sample preparation strategy. By minimizing matrix interference and maximizing analyte recovery, LLE ensures enhanced sensitivity, reproducibility, and precision, solidifying its selection as the preferred method for the robust bioanalytical quantification of resveratrol isomers in biological fluids.

1. Comparison of Chromatographic Performance for the Analysis of trans-RES and cis-RES in Plasma by using Protein Precipitation (PPT) and Liquid–Liquid Extraction (LLE) Methods .

parameters protein precipitation (PPT) liquid–liquid extraction (LLE)
retention time (R t) trans-RES 4.368 ± 0.085a 4.418 ± 0.032a
retention time (R t) cis-RES 5.768 ± 0.04b 5.678 ± 0.02b
resolution (R s) trans-RES vs cis-RES 6.10 ± 0.045c 7.85 ± 0.20d
number of theoretical plates (NTP) 11843 ± 100.80e 13650 ± 127.89f
tailing factor (T f) 0.93 ± 0.001g 1.001 ± 0.004g
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Data are mean values ± standard deviation (n = 3). Significant differences between methods (PPT and LLE) for each parameter were determined using an independent t-test at a 5% level of significance (p < 0.05). The extraction volume ratio for both methods was 1:10 (plasma/extraction solvent).

Optimization of Chromatographic Conditions

The effect of mobile phase composition 0.05% Formic in water/acetonitrile on chromatographic performance was evaluated using ratios of 50:50, 60:40, and 70:30 (v/v) (Table ). An increase in acetonitrile content from 50% to 70% resulted in a progressive reduction in the retention time (R t) of trans-RES from 7.870 ± 0.020 min (70:30) to 3.250 ± 0.025 min (50:50), consistent with reversed-phase chromatographic theory, where higher organic solvent content enhances elution strength and accelerates analyte migration.

2. Chromatographic Parameters of trans-RES under Different Eluent Conditions .

  0.05% formic acid in water/ACN (v/v)
parameter (50:50) (60:40) (70:30)
retention time (R t, min) trans-RES 3.250 ± 0.025 4.447 ± 0.042 7.870 ± 0.020
retention time (R t) cis-RES 5.440 ± 0.50 6.310 ± 0.20 9.035 ± 0.045
peak area trans-RES 6255 ± 50.55 6546 ± 37.63 6345 ± 45.45
peak area internal standard 77.3 ± 20.45 77.5 ± 10.45 76.8 ± 35.20
peak area ratio (PAR) 80.92 ± 10.05 83.94 ± 30.45 82.62 ± 40.45
resolution (R s) 5.610 ± 0.25 7.410 ± 0.40 6.811 ± 0.10
tailing factor (T f) 0.490 ± 0.11 1.01 ± 0.004 1.351 ± 0.001
number of theoretical plates (NTP) 10101 ± 127.69 13650 ± 127.89 12830 ± 100.20
height equivalent to a theoretical plate (HETP) 0.002 0.002 0.002
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Data are mean values ± standard deviation (n = 3).

Critically, the resolution (R s) between geometric isomers trans-RES and cis-RES was significantly influenced by the mobile phase composition. The separation of these isomers is particularly challenging due to their similar chemical properties. The R s value, which is the definitive metric for assessing the degree of separation between two adjacent peaks, was highest at the 60:40 composition (7.410 ± 0.40). This value far exceeds the minimum requirement for baseline resolution (R s ≥ 1.5), , indicating an excellent separation where the two compounds are fully resolved from one another, thereby minimizing integration errors and ensuring accurate individual quantification. In contrast, the R s values for the 50:50 (5.610 ± 0.25) and 70:30 (6.811 ± 0.10) ratios were still acceptable for baseline separation but were markedly lower. The superior resolution at the 60:40 ratio can be attributed to an optimal balance of elution strength and selectivity, allowing for sufficient differential interaction of each isomer with the stationary phase. , The calculated relative retention (α) was also the highest at this ratio, further confirming enhanced selectivity for the pair.

The highest peak area for trans-RES was observed at the 60:40 ratio (6546 ± 37.63), suggesting the optimal detector response and solvent interaction at this composition. Peak Area Ratio (PAR) values were comparable across compositions, indicating consistent quantification relative to the internal standard. The tailing factor (T f) was also optimal at 0.93 ± 0.004 for the 60:40 ratio, well within the acceptable range (0.8–2.0), which ensures peak symmetry and reliable integration. Column efficiency, as measured by the number of theoretical plates (NTP), was highest at the 60:40 ratio (13,650 ± 127.89), while the height equivalent to a theoretical plate (HETP) remained constant across all conditions. Collectively, these findings demonstrate that the 60:40 (v/v) 0.05% formic acid in the acetonitrile-to-aqueous phase ratio provides the best overall chromatographic performance. It achieves an ideal balance, offering superior resolution between the critical pair of trans- and cis-RES, excellent peak symmetry, high column efficiency, and an optimal analysis time, as supported by the chromatogram in Figure C.

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Chromatogram profiles of the plasma blank (A), internal standard in plasma (B), and resveratrol standard in plasma (C).

Full Validation Results for Human Plasma Analysis

Specificity

The specificity of the analytical method was confirmed by evaluating the chromatograms of three different samples. The chromatogram profile of the plasma blank (Figure A) showed no interference peaks at the same retention time as trans-RES and the internal standard, proving that endogenous components in the plasma matrix did not interfere with the analysis. Furthermore, the plasma chromatogram supplemented with the internal standard (Figure B) showed sharp, symmetrical peaks at the internal standard’s retention time, without interference, confirming its suitability as a reference. Most importantly, the plasma chromatogram supplemented with a resveratrol standard and an internal standard (Figure C) shows two well-separated peaks and perfect resolution, with the trans-RES and cis-RES isomer peaks clearly detected without interference from the plasma matrix. The specificity test results confirm that the developed method has high selectivity and can specifically identify trans-RES and cis-RES in the plasma matrix, thereby meeting the requirements for application in pharmacokinetic studies as outlined in the ICH guideline.

Linearity

Sequential quantitative dilutions of the resveratrol stock solution were used to establish linearity at nine concentration levels, ranging from 0.025 to 50 μg/mL. The chromatographic response was recorded, and the concentration of the standard solution was plotted against the average peak area (Figure ), with the X-axis representing the concentration and the Y-axis representing the peak area response. The analytical method, using a mobile phase mixture of 0.05% formic acid in Water/ACN (60:40, v/v) at a flow rate of 1 mL/min and a column temperature of 30 °C, showed excellent linearity with a regression equation of y = 1.679x + 0.40 and an R 2 of 0.9999 for the human plasma calibration curve. This R 2 value exceeds the ICH M10 requirement of a minimum of 0.997 for bioanalytical validation. , The percentage coefficient of variation (%CV) for all calibration points is shown in Table S1. It is within the acceptable limit of ± 15%, except for the lower limit of quantification (LLOQ), where a limit of ± 20% is permitted. ,

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Linear calibration curve of trans-RES by RP-HPLC using human and rat plasma.

Sensitivity

Lower limit of quantification (LLOQ) for trans-RES in this study was established at 0.025 μg/mL, in full compliance with the precision and accuracy criteria outlined in the ICH M10 guideline. The detailed validation data supporting this LLOQ are provided in Table S2. At 0.025 μg/mL, the mean accuracy, expressed as %CV from the nominal concentration, was well within the acceptable range of ± 20%, with an observed deviation of only ± 3.65%. This demonstrates the excellent reliability and robustness of the LLOQ for routine quantification. Compared with existing methodologies, the LLOQ achieved in this study exhibits competitive sensitivity. For instance, Rahaman et al. 2024 reported an LLOQ of 0.28 μg/mL for trans-RES, while Jagwani et al. 2020 developed an RP-HPLC method with an LLOQ of 0.030 μg/mL for trans-RES in human plasma. The significantly lower LLOQ of 0.025 μg/mL achieved herein underscores the enhanced sensitivity of the present method. This high level of sensitivity makes the proposed method a reliable and robust analytical tool for quantifying trans-RES in plasma, particularly in complex biological matrices where low analyte concentrations are expected.

Precision and Accuracy

Validation of the developed HPLC method demonstrated excellent precision, with intraday and interday precision (%CV) consistently below 5% at all QC levels 0.075 μg/mL (LQC), 25 μg/mL (MQC), and 37.5 μg/mL (HQC), surpassing the ≤ 15% acceptance criteria specified in ICH M10 guideline. The results in Table align with bioanalytical theory, emphasizing that low %CV values reflect minimal random error and high method robustness. , The accuracy, assessed via recovery studies, ranged from 93.90% to 98.19%, consistently adhering to the ICH-recommended 80–120% (LQC) and 85–115% (MQC and HQC) ranges. The high recovery efficiency corroborates theoretical principles of extraction optimization, where effective analyte isolation from biological matrices minimizes matrix effects and maximizes detection fidelity. , This assertion is further substantiated by matrix effect evaluations, which showed values ranging from 97.22% to 102.80% for trans-RES and 96.84% to 98.50% for the internal standard (IS), both within the acceptable ± 15% range stipulated by the guideline. , This outcome corroborates the absence of significant ionization suppression or enhancement. These results validate that the liquid–liquid extraction procedure effectively reduces matrix interferences, thereby ensuring method specificity, accuracy, and suitability for the precise quantification of trans-RES in complex biological matrices. The selection of carbamazepine as the IS was based on its physicochemical similarity to trans-RES, including a comparable Log P (2.45), which promotes consistent extraction efficiency during LLE. , Chromatographically, carbamazepine elutes at a distinct retention time (6.2 min), fully resolved from both trans-RES (4.4 min) and cis-RES (5.7 min) without interference, further confirming its reliability as an internal standard.

3. Method Validation Results: Precision and Accuracy for Quantification of trans-RES in Human Plasma .
    precision
 accuracy
 matrixes effect
intraday
 interday
 extracted
 non extracted
 recovery (%)
        
trans-res (μg/mL) day avg (μg/mL) %CV avg (μg/mL) %CV avg (μg/mL) avg (μg/mL) trans-res %CV trans-res %CV) IS %CV
0.075 1 0.08 3.419 0.083 3.096 0.079 0.085 93.90 2.8 97.22 6.84 98.50 4.45
2 0.083 2.376
3 0.085 0.536
25 1 26.03 1.823 25.841 1.97 23.24 23.67 98.19 1.00 102.80 2.61 96.80 0.87
2 25.264 0.785
3 26.229 4.526
37.5 1 38.278 1.356 37.343 2.882 40.32 41.68 96.77 3.05 99.45 2.98 97.65 3.25
2 36.167 3.264
3 37.584 3.444
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Data are mean values ± standard deviation (n = 3).

Evaluation Stability of trans-RES in Human Plasma

Stability of trans-RES in human plasma was comprehensively evaluated in triplicate at LQC and HQC levels, covering analyzing the effects of three freeze–thaw cycles, short-term storage at room temperature (25 °C), long-term storage at −20 °C, and postpreparative stability in the autosampler for 24 h, as shown in Table Samples were considered stable when the mean concentration demonstrated accuracy within ± 15% deviation from the nominal value and precision within ± 15% CV. All stability results for trans-RES fell well within these predefined acceptance criteria, confirming the compound’s excellent stability under routine handling, processing, and storage conditions. The reliability of the bioanalytical method for pharmacokinetic studies was thereby verified.

4. Stability of the trans-RES in the Matrix under Various Storage Conditions .
stability condition level measured conc. (μg/mL) %CV
freeze–thaw stability in matrix after three cycles at −25°C LQC 0.080 ± 0.005 6.348
HQC 37.931 ± 0.636 1.677
short-term (benchtop) stability in matrix 25°C during 12 h LQC 0.079 ± 0.002 2.227
HQC 40.231 ± 0.878 2.183
25°C during 24 h LQC 0.079 ± 0.003 3.199
HQC 37.869 ± 1.164 3.073
long-term stability in matrix –20°C during 7 Days LQC 0.081 ± 0.002 2.727
HQC 37.634 ± 0.775 0.356
–20°C during 14 Days LQC 0.080 ± 0.005 6.043
HQC 36.750 ± 0.285 2.058
–20°C during 30 Days LQC 0.076 ± 0.024 6.744
HQC 36.54 ± 0.432 2.456
autosampler 25°C during 48 h LQC 0.079 ± 0.005 6.539
HQC 36.774 ± 0.289 0.787
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Data are mean values ± standard deviation (n = 3).

Photostability of trans-RES Blood Plasma

Based on the chromatographic profile in Figure , trans-RES proved to be sensitive to light in the plasma matrix, as indicated by a progressive decrease in the trans peak and the appearance of the cis-RES peak during the 30 min incubation. At 0 min, the trans peak was sharp and intense. − Then, after 30 min, its intensity decreased while a new peak, identified as cis-RES, appeared. This finding is consistent with the photoisomerization of stilbene under UV illumination (365 nm, UVA), where excitation to an excited state allows rotation around the central CC bond and conversion from trans to cis. − In this context, UV light is the main factor in isomerization. − Quantitative analysis of the isomer ratio revealed a clear kinetic trend (Table S4). While trans-RES remained at 100% for the first 15 min, its proportion dropped to 81.97% at 30 min, 77.36% at 45 min, and 58.57% after 60 min of exposure. Conversely, cis-RES emerged only after 30 min (18.03%) and increased to 22.64% and 41.43% at 45 and 60 min, respectively. The photoisomerization from trans- to cis-RES followed apparent first-order kinetics from 30 to 60 min of exposure, with a rate constant (K e) of 0.67 h–1 and a corresponding half-life (t 1/2) of approximately 62 min under the experimental conditions. This quantitative kinetic parameter underscores the rapid photodegradation of trans-RES in plasma and reinforces the necessity for stringent light protection during sample handling and analysis.

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HPLC chromatograms of trans-RES in plasma following UVA (365 nm) exposure for different time intervals (0–60 min).

The plasma pH was monitored throughout the light-exposure experiment and remained within the physiological range (7.39 at 0 min, 7.39 at 5 min, 7.40 at 15 min, 7.41 at 30 min, and 7.45 at 60 min). It is known that physiological plasma pH can modulate photochemical reactivity by partially deprotonating, forming phenoxide anions (-O–). The negative charge increases the electron density on the CC double bond, stabilizing the transition state and lowering the activation energy. As a result, the conversion rate of trans-RES to cis-RES increases significantly. This observation has analytical implications, such as light exposure reducing the trans-RES signal, causing secondary peaks from isomers and degradation products, and risking bias in quantification. Therefore, sample handling and analysis must minimize photochemical stress. Implementing these controls improves detection sensitivity, maintains peak integrity, and ensures the reliable quantification of trans-RES in plasma.

Partial Validation Results for Rat Plasma Analysis

After a full validation in human plasma was completed, a partial validation was conducted in rat plasma to evaluate method suitability for preclinical studies. As summarized in Table , the method demonstrated exemplary performance across all validation parameters, in accordance with the international bioanalytical validation method guideline. The method exhibited excellent linearity over the concentration range of 0.025–50 μg/mL, with a correlation coefficient (R 2) > 0.998. The calibration curve obtained from rat plasma analysis yielded a regression equation of y = 1.659x + 0.034 (Figure ), demonstrating a consistent and proportional response across the validated range.

5. Results of Partial Validation Testing of Bioanalytical Methods for the Quantification of trans-RES in Rat Plasma.
parameter value
precision (CV, intraday/interday) 2.39/4.18 at 0.075 μg/mL
3.29/1.66 at 25 μg/mL
4.95/3.66 at 37.5 μg/mL
accuracy (% recovery) 97.90 at 0.075 μg/mL
97.49 at 25 μg/mL
96.19 at 37.5 μg/mL
matrix effect (ME) (%) 96.5 ± 2.56%–103.1 ± 4.33%
tailling factor (T f) 1.002 ± 0.002
lower limit of quantification (LLOQ) 0.025 μg/mL
number of theoretical plates (NTP) 13002 ± 100.08
height equivalent to a theoretical plate (HETP) 0.0019
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The method’s precision was outstanding, with intra- and interday %CV significantly below the 15% acceptance criterion at all concentration levels, including the LQC level (0.075 μg/mL), indicating excellent reliability and reproducibility over time. Furthermore, a critical indicator of instrumental precision and chromatographic performance, the system suitability test was rigorously assessed through six replicate injections of a standard solution (Table S3.). The resulting %CV for the analyte peak area and retention time was well below 15%, confirming a stable and robust chromatographic system throughout the validation runs. This consistency ensures data integrity and aligns with the observed method precision. Method accuracy, reflected by recovery values ranging from 96.19% to 97.90%, demonstrates high accuracy with respect to the actual values and confirms optimal extraction efficiency with adequate selectivity against rat plasma matrix interference. Evaluation of matrix effect, critical for assessing ion suppression or enhancement, showed values between 96.5% and 103.1%, well within the accepted range of 85–115%. These results indicate negligible matrix interference and confirm that the liquid–liquid extraction procedure effectively removes endogenous phospholipids and other plasma components, thereby ensuring consistent analyte ionization and reliable quantification of trans-RES in rat plasma. The method exhibits remarkable sensitivity, with an LLOQ of 0.025 μg/mL, enabling reliable quantification at very low concentrations, a critical requirement for comprehensive pharmacokinetic characterization. Chromatographic integrity was demonstrated by excellent peak symmetry (T f = 1.002) and high column efficiency, as evidenced by an NTP of approximately 13002 ± 100.08 and a minimal HETP of 0.0019, ensuring optimal resolution and accurate peak integration. ,

The sample preparation protocol proved to be efficient and reproducible, yielding consistently high recovery rates. The validation was conducted in accordance with the ICH M10 guideline. Comprehensive validation of human and rat plasma matrices confirmed that the method meets all of the predetermined acceptance criteria. The successful partial validation, supported by the stringent system suitability results, demonstrates the method’s robustness and transferability between species, strongly supporting its application in regulatory submissions for preclinical development studies.

Pharmacokinetic Study

A validated HPLC method was successfully applied to characterize the comprehensive pharmacokinetic profile of trans-RES in rat plasma following administration via five different routes, as shown in Figure and revealed highly significant differences (p < 0.05) in the disposition profile of the compound among the evaluated administration routes, as demonstrated by Tukey’s posthoc test results Table . These differences quantitatively illustrate the fundamental influence of route-specific physiology on the drug’s disposition in the body and confirm that systemic exposure and disposition kinetics of trans-RES are highly dependent on the route of administration, a characteristic of compounds that are susceptible to extensive presystemic metabolism.

5.

5

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Plasma concentration–time profile of trans-RES in rats following administration via various routes (PO, SC, IM, IP, and IV) over 24 h.

6. Pharmacokinetic Parameters of trans-RES Following Administration on Different Routes in Rats .
  route
parameter PO SC IM IP IV
slope (k) (h–1) 0.1911 ± 0.0069 0.2547 ± 0.0031 0.2640 ± 0.0067 0.3085 ± 0.0076 0.5361 ± 0.0204
AUC0–t (ng·h·mL–1) 187.82 ± 12.33a 1295.49 ± 40.57b 1072.54 ± 36.96c 3279.91 ± 125.80d 3866.02 ± 75.14e
AUC0–∞ (ng·h·mL–1) 189.97 ± 12.18a 1299.13 ± 40.76b 1074.62 ± 37.01c 3283.07 ± 125.57d 3866.15 ± 75.19e
AUCt–∞ (ng·h·mL–1) 2.148 ± 0.161a 3.644 ± 0.248b 2.085 ± 0.330a 3.152 ± 0.310a,b 0.131 ± 0.048c
C max (ng·mL–1) 92.34 ± 5.78a 245.28 ± 3.49b 367.17 ± 0.85c 1143.78 ± 10.41d 1386.43 ± 6.88e
t max (h) 0.17 ± 0.00a 0.33 ± 0.00b 0.17 ± 0.00b 0.17 ± 0.00b 0.08 ± 0.00c
t 1/2 (h) 3.63 ± 0.13a 2.72 ± 0.03b 2.63 ± 0.07b,c 2.25 ± 0.06c 1.29 ± 0.05d
C L (L·h–1) 42.2275 ± 2.683a 6.1620 ± 0.1952b 7.4504 ± 0.2558b 2.4391 ± 0.0918c 2.0698 ± 0.0402c
V z (L) 221.42 ± 21.49a 24.20 ± 0.88b 28.24 ± 1.16b 7.91 ± 0.47c 3.86 ± 0.07d
F abs (%) 4.91 ± 0.22a 33.60 ± 1.03b 27.80 ± 0.42b 84.92 ± 4.87c 100 ± 0.00d
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a

Values are presented as mean ± SD. Different superscript letters (a, b, c, d, e) within the same row indicate a statistically significant difference (p < 0.05) based on Tukey’s HSD posthoc test. Shared letters indicate no significant difference between routes.

The intravenous (IV) route yielded the highest systemic exposure (AUC0–∞) and peak concentration (C max), at 3866.15 ng.h.mL–1 and 1386.43 ng.mL–1, respectively, which were significantly higher (p < 0.05) than those for all other routes. The time to peak concentration (T max) was also the fastest (0.08 h). The low volume of distribution (V z ) (3.86 L) indicates that the formulation was primarily distributed in the central compartment (blood and extracellular fluid), consistent with its 100% absolute bioavailability serving as the reference. Among the non-IV routes, intraperitoneal (IP) administration resulted in the highest AUC0–∞, C max, and absolute bioavailability (F abs) at 3283.07 ng.h.mL–1, 1143.78 ng.mL–1, and 84.92%, respectively. These values were significantly higher than those for the SC, IM, and PO routes but still lower than those for IV. This high bioavailability is attributed to the large absorptive surface area of the peritoneal cavity and lymphatic drainage, which may allow a portion of the drug to bypass first-pass hepatic metabolism. IP absorption was rapid (T max = 0.17 h), similar to the PO and IM routes, but significantly different from SC (T max = 0.33 h). Meanwhile, the subcutaneous (SC) and intramuscular (IM) routes showed pharmacokinetic profiles that were significantly different from those of IV, IP, and PO. However, several parameters between SC and IM were not significantly different. The clearance (C L) for both routes was not significantly different (6.16 and 7.45 L.h–1, respectively), yet both were significantly lower than PO and higher than IP and IV. Their volumes of distribution were also not significantly different (24.20 and 28.24 L), indicating comparable tissue penetration, and both were significantly smaller than PO but larger than IP and IV. The bioavailability of both routes (33.60% for SC and 27.80% for IM) was also not statistically different. These findings reflect the complex interplay between tissue vascularization, blood flow rate, and formulation properties at the injection site. , Oral administration (PO) yielded the least favorable kinetics. The parameters AUC0–∞, C max, and F abs (4.91%) were significantly lower than those for all routes. Conversely, the volume of distribution and clearance were significantly the highest (221.42 and 42.23 L.h–1). This indicates that despite relatively rapid initial absorption (T max = 0.17 h), the drug undergoes extensive first-pass metabolism in the liver following gastrointestinal absorption, drastically reducing the fraction that reaches systemic circulation. , The large volume of distribution signifies extensive distribution into peripheral tissues, likely due to trans-RES lipophilic nature. These findings are consistent with previous reports showing low oral bioavailability in rat models (6%) and even lower in humans (<1%). ,

This comparative analysis confirms the occurrence of flip-flop kinetics, particularly following oral administration, as illustrated in Figure . To provide quantitative evidence, the absorption rate constant (K a) was estimated for each nonintravenous route using a one-compartment open model with first-order absorption, fitted to the mean plasma concentration–time profiles. The true elimination rate constant (K e), derived from intravenous data, was 0.5361 ± 0.0204 h–1, corresponding to a half-life (t 1/2) of 1.29 h (Figure A). The estimated K a values for non-IV routes were significantly lower at 0.1911 ± 0.0069 h–1 for oral (PO), 0.2547 ± 0.0031 h–1 for subcutaneous (SC), 0.2640 ± 0.0067 h–1 for intramuscular (IM), and 0.3085 ± 0.0076 h–1 for intraperitoneal (IP). Crucially, K a was less than K e for all extravascular routes, with K a/K e ratios of 0.36 (PO), 0.48 (SC), 0.49 (IM), and 0.58 (IP). This consistent relationship quantitatively verifies that absorption is slower than elimination, making it the rate-limiting step in the pharmacokinetic process. , Consequently, the terminal phase of the concentration–time profile reflects slow absorption kinetics rather than actual elimination, leading to the observed prolongation of the apparent half-life (t 1/2 = 2.25–3.63 h; Figure B) compared with the actual elimination half-life (1.29 h). This numerical evidence robustly validates flip-flop kinetics for trans-RES. It underscores the imperative of formulating strategies to enhance absorption rates, improve bioavailability, and ensure accurate interpretation of pharmacokinetic parameters.

6.

6

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Pharmacokinetic flip-flop scheme of trans-RES: effect of administration route (IV vs Non-IV) on half-life (t 1/2) and plasma concentration profile.

In order to support such formulation development and enable reliable pharmacokinetic characterization across diverse administration routes, a robust, accessible, and cost-effective bioanalytical method is indispensable. A direct comparative analysis of the developed method against previously reported assays (Table ) underscores its strategic advantages, especially for resource-constrained settings. The RP-HPLC-UV method under consideration exhibits a strategic balance between the performance and practicality. While liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods offer superior sensitivity (LLOQ 5–6 ng/mL), they require substantially higher instrumentation costs and specialized technical expertise, creating significant barriers for resource-limited laboratories. , In contrast, our method demonstrates sensitivity (LLOQ = 25 ng/mL) comparable to that of other HPLC-UV methods while offering several distinct advantages. In terms of operational efficiency, our isocratic method (11 min of runtime) provides faster analysis than the gradient HPLC methods (15 min) reported by Jagwani et al. and Gadag et al. , This enables higher sample throughput without compromising the resolution.

7. Comparison of Method Validation Parameters, Analytical Performance, and Greenness Assessment of Various Bioanalytical Methods for the Determination of trans-RES in Biological Matrices__________________.

graphic file with name ao5c12760_0007.jpg

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Furthermore, the method incorporates comprehensive photostability assessment and full validation according to the ICH M10 guideline features often absent in earlier reports. It was successfully applied in a comparative pharmacokinetic study across five administration routes. A notable limitation of this study is the modest sample size (n = 3 per route), which may compromise the precision of the estimated pharmacokinetic parameters, particularly for the highly variable oral route. Future studies would benefit from a larger cohort to improve the statistical power and generalizability of the findings. Notwithstanding, the method has been demonstrated to be a sensitive, cost-effective, and readily implementable solution, thereby rendering robust trans-RES quantification accessible for formulation and pharmacokinetic research across academic, industrial, and clinical laboratories.

Analytical GREEnness (AGREE)

Within the framework of Green Analytical Chemistry (GAC), the environmental sustainability of the developed RP-HPLC-UV method was quantitatively assessed using the Analytical GREEnness (AGREE) metrics. This tool evaluates a method’s compliance with the 12 principles of green analytical chemistry. The assessment yielded a score of 0.7 on a 0–1 scale, which is visualized in a colored circular diagram divided into sectors, each representing one principle. , A score of 0.7 indicates that the method possesses favorable and environmentally friendly attributes. The green advantages of this method become particularly evident when compared with those of other referenced methods. In terms of instrumentation, the use of HPLC-UV offers superior energy efficiency compared to techniques such as LC-MS/MS or UHPLC-Q-Orbitrap MS/MS, which require high energy consumption for ionization, vacuum generation, and system cooling. ,

The application of an isocratic mobile phase (ACN/H2O + 0.05% FA) significantly reduces the volume and complexity of organic solvent use compared with gradient elution. The relatively short analysis time (7.5 min) further contributes to energy and material conservation. During sample preparation, although the liquid–liquid extraction method with ethyl acetate still uses organic solvents, the volume used is controlled, and the solvent is relatively safer than alternatives such as MTBE. The high and consistent recovery rate (93.90–98.19%) and excellent resolution (R s = 7.85) between the trans- and cis-RES isomers help minimize the risk of repeat analyses. This ultimately prevents the waste of reagents and energy. Therefore, this method offers an ideal balance between rigorous analytical performance and sustainability principles, making it a greener and more accessible choice compared to more resource-intensive LC-MS/MS methods and more efficient than several other HPLC-UV methods that employ a gradient approach.

Conclusions

This study describes the development and validation of a reliable RP-HPLC-UV method for quantifying trans-RES in plasma and assessing the integrity of its isomers under the stress conditions. The developed method complies with the ICH M10 guideline, demonstrating excellent linearity (0.025–50 μg/mL; R 2 ≥ 0.999), high precision (%CV < 5%), accuracy (93.90–98.19%), and high sensitivity (LLOQ 0.025 μg/mL). Stability testing confirmed the robustness of the procedure under routine handling but also revealed significant photoisomerization upon UVA exposure, underscoring the necessity of light-protected protocols throughout analysis. Pharmacokinetic studies across five administration routes provided a comprehensive comparative profile, revealing low oral bioavailability (4.91 ± 0.22%), high intraperitoneal bioavailability (84.92 ± 4.87%), and flip-flop kinetics for all nonintravenous routes. To the best of our knowledge, this is the first study to compare trans-RES pharmacokinetics across five routes using a fully validated RP-HPLC method incorporating photostability controls. Furthermore, the environmental sustainability of the method was affirmed through the Analytical Greenness (AGREE) assessment, yielding a score of 0.7, reflecting favorable green attributes such as energy-efficient isocratic elution, minimized solvent consumption, and reduced waste generation. Collectively, this method offers a reliable, cost-effective, and environmentally considerate platform for routine bioanalysis, while the pharmacokinetic insights provide a valuable foundation for formulation strategies to enhance the bioavailability and therapeutic potential of trans-RES.

Supplementary Material

ao5c12760_si_001.pdf (99.3KB, pdf)

Acknowledgments

The authors acknowledge the facilities and scientific and technical support from Advanced Characterization Laboratories in Serpong, National Research and Innovation Agency (BRIN), through the E-Science Service, as well as the Institute of Tarumanagara for providing access to HPLC instrumentation and supporting facilities that contributed to this research.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12760.

  • Linear calibration curves of trans-resveratrol in human and rat plasma by RP-HPLC; lower limit of quantification (LLOQ) data for trans-RES; accuracy, precision, and system suitability results for trans-RES quantification; and percentage of trans; and cis-RES in plasma during UVA (365 nm) exposure at different time intervals (PDF)

A.M.: conceived and designed the experiments, performed the experiments, analyzed and interpreted data, and wrote the paper. M.J.: conceived and designed the experiments, and analyzed and interpreted data. Witta K.R.: conceived and designed the experiments, and analyzed and interpreted data. N.B. M.H.: conceived and designed the experiments, and analyzed and interpreted data. A.B.: conceived and designed the experiments, contributed reagents and materials, performed the experiments, analyzed and interpreted data, and wrote the paper.

The authors are grateful for the funding provided by Hibah PUTI Quartile 1 2025, Number: NKB-178/UN2.RST/HKP.05.00/2025.

The authors declare no competing financial interest.

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