<p><strong>This study presents an optimized dispersive liquid–liquid microextraction (DLLME) method coupled with GC-FID for the determination of eight priority polycyclic aromatic hydrocarbons in surface waters. Experimental design approaches, including fractional factorial and Box–Behnken designs, were used to optimize extraction conditions and maximize analytical performance.</strong></p><p>The validated method achieved rapid chromatographic separation, low detection limits, good precision, and high enrichment factors while maintaining favorable green analytical characteristics. Successful application to wastewater, lake, river, and drain water samples demonstrates its suitability for routine environmental monitoring of PAHs.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489"><strong>Experimental Design-Based Dispersive Liquid–Liquid Microextraction with GC-FID for Determination of Polycyclic Aromatic Hydrocarbons in Surface Water</strong></a></h4><p>Chen Minghong, Song Peiyu, Tien Ping Lee, Garret Chan Zhe Ming, Philip J. Marriott, and Yong Foo Wong*</p><p>ACS Omega 2026, 11, 21, 31418–31431</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.6c01489">https://doi.org/10.1021/acsomega.6c01489</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Polycyclic aromatic hydrocarbons (PAHs) are organic pollutants containing two or more fused benzene rings and are widely associated with carcinogenic, teratogenic, and mutagenic properties. <a href="javascript:void(0);">(1)</a> These hydrophobic substances can bioaccumulate with long environmental half-lives and are classified as high priority ecotoxicants of the first danger class. <a href="javascript:void(0);">(1,2)</a> Anthropogenic activities (e.g., fossil fuel combustion, industrial processes, biomass burning, and others) are predominantly recognized as the primary sources of PAHs in most environmental compartments. <a href="javascript:void(0);">(3−6)</a> In the present work, the hydrophobic nature of PAHs facilitates adsorption onto suspended particulate matter and organic material in aquatic systems, followed by deposition into underlying sediments. <a href="javascript:void(0);">(7)</a> These persistent organic pollutants have been reported in surface waters as well as estuarine and coastal environments and can potentially disrupt ecosystem functioning. <a href="javascript:void(0);">(7,8)</a></p><p>From a public health perspective, PAHs exposure via upon ingestion, has been linked to a spectrum of adverse health effects such as cancer and reduced fertility. <a href="javascript:void(0);">(2,9)</a> Sixteen unsubstituted PAHs have been designated as priority pollutants by the United States Environmental Protection Agency (USEPA). <a href="javascript:void(0);">(10)</a> The International Agency for Research on Cancer (IARC) has classified benzo(a)pyrene (BaP) in Group 1 (carcinogen to humans) and other PAHs in Group 2A (possible human carcinogens). <a href="javascript:void(0);">(10,11)</a> The maximum permissible concentrations of benzo(a)pyrene (0.00001 mg/L) in water intended for drinking water is regulated by the European Union (EU). The EU limits the total content of polyaromatic compounds in drinking water to &lt;0.1 μg/L. <a href="javascript:void(0);">(12)</a> In heavily polluted waterways, PAH concentrations have been reported to reach 10 μg/mL. <a href="javascript:void(0);">(13)</a> Epidemiological evidence also suggests a positive relationship between increased cancer incidence in human populations that are having high exposure to organic ecotoxins. <a href="javascript:void(0);">(14)</a> For these reasons, quantitative assessments of PAH occurrence and distribution in surface waters remain important and fertile research topics. <a href="javascript:void(0);">(15,16)</a></p><p>PAHs in water and environmental matrices are routinely analyzed using either gas chromatography (GC) and/or liquid chromatography (LC) with various detection modalities. <a href="javascript:void(0);">(17,18)</a> The coupling of chromatography to mass spectrometry has been the preferred technique, owing to the distinct selectivity and sensitivity, particularly when operated in selected ion monitoring (SIM) or multiple reaction monitoring (SRM) modes. <a href="javascript:void(0);">(19)</a> Additionally, LC coupled with fluorescence detection is also extensively utilized for PAH analysis. <a href="javascript:void(0);">(20,21)</a> As PAHs typically occur at trace to ultratrace levels in complex real-world matrices, sample pretreatment and/or preconcentration is generally required prior to instrumental analysis. <a href="javascript:void(0);">(22,23)</a> Conventional approaches such as liquid–liquid extraction and solid-phase extraction are widely used but are often solvent-intensive and laborious and generate substantial hazardous waste. More recently, microextraction techniques (e.g., dispersive microsolid phase extraction, dispersive liquid–liquid microextraction (DLLME), solid phase microextraction, etc.) have emerged as attractive alternatives, providing advantages such as speed and simplicity and requiring substantially lower volumes of hazardous solvents. <a href="javascript:void(0);">(24−26)</a></p><p>DLLME has shown good efficiency for extracting and preconcentrating hydrophobic PAHs from aqueous matrixes. <a href="javascript:void(0);">(24,27)</a> Briefly, DLLME involves rapid injection of a small volume of an extraction solvent (immiscible with water) and dispersive solvent into the aqueous sample. <a href="javascript:void(0);">(24)</a> Target analytes partition into the microdroplets and are subsequently recovered in a minimal volume of the organic phase after centrifugation. The large interfacial contact area between the two phases enhances mass transfer, leading to fast attainment of extraction equilibrium with high enrichment factors. <a href="javascript:void(0);">(28,29)</a> DLLME has been applied to a wide range of matrices, including environmental samples, <a href="javascript:void(0);">(25,30)</a> milk and dairy products, <a href="javascript:void(0);">(31)</a> honey, <a href="javascript:void(0);">(32)</a> and others. <a href="javascript:void(0);">(33)</a> Optimization of DLLME conditions for targeted solutes is often performed using a one-factor-at-a-time approach, which is time-consuming, labor-intensive, and may inadequately take into account interaction between or among variables. <a href="javascript:void(0);">(34)</a> In contrast, multivariate experimental design methodologies accommodate simultaneous variation of multiple extraction variables, facilitating identification of interaction effects and development of statistically robust models. <a href="javascript:void(0);">(35−37)</a></p><p>Over the past decade, multivariate design of experiments (DOE) has become an important optimization strategy for process development. Compared with traditional one-factor-at-a-time approaches, DOE can reduce time, effort, and resource consumption while extracting meaningful information from a relatively small number of experimental analyses. <a href="javascript:void(0);">(37−40)</a> Among the DOE frameworks, the Box-Behnken design (BBD), an economical variant of response surface methodology, has been widely used for process optimization, offering high statistical efficiency and good predictive performance when tuning experimental variables in analytical and industrial applications. <a href="javascript:void(0);">(35,39,41−45)</a> This study evaluates the applicability of using RSM to optimize DLLME conditions for selective extraction and preconcentration of acenaphthene, fluorene, anthracene, phenanthrene, fluoranthene, pyrene, benzo(a)anthracene, and benzo(a)pyrene. A fractional factorial design (FrFD) was initially used to screen and identify statistically significant DLLME variables. The selected factors were then optimized using the BBD to construct quadratic models and generate response surface plots for determining optimal extraction conditions. The optimized DLLME-GC-FID method was validated, and AGREEprep and AGREE were used to quantify ‘greenness’ metric scores. Analytical practicability of the validated method was demonstrated by determining the targeted PAHs in several surface water samples.</p><h4><strong>2. Experimental Section</strong></h4><h5><strong>2.4. GC-FID Analysis</strong></h5><p>All experiments were performed using an <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=7890B"><strong>Agilent 7890B GC system</strong></a> equipped with a <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library?mainauthorItems=10&amp;search=7693A"><strong>G4513A autosampler</strong></a> and a split–splitless inlet (Agilent Technologies; Santa Clara, CA, USA). Chromatographic separation was effected by using a <strong>DB-5HT column</strong> (Agilent Technologies, Santa Clara, CA, USA; 30 m × 0.25 mm I.D. × 0.25 μm film thickness). Injection mode and oven temperature programming were optimized to achieve baseline separation of all target PAHs. Helium (99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min. Splitless mode with a splitless time of 2.1 min was used, with a 1 μL injection volume. The injector temperature was maintained at 300 °C. The oven temperature program was 70 °C (hold for 3.3 min) and ramped to 300 °C (hold 5 min) at a rate of 25 °C/min. The FID temperature was set to 300 °C.</p><h4><strong>3. Results and Discussion</strong></h4><p>The initial GC-FID conditions were adopted from Hor et al. for the determination of BAP, BAA, BBF, and chrysene in tocotrienol concentrates. <a href="javascript:void(0);">(52)</a> Under those chromatographic conditions, baseline separation of all the targeted PAHs could not be achieved (Flt, Pyr, BaA, and BaP). Hence, GC conditions, particularly the oven temperature program, were briefly optimized to obtain baseline separation of all the targeted PAHs (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489#fig1">Figure 1</a>A).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_21_31418_31431_Figure_1_Chromatograms_of_Ai_standard_mixture_265d7e1014.jpeg" alt="ACS Omega 2026, 11, 21, 31418–31431: Figure 1. Chromatograms of (Ai) standard mixture (100 μg/L) before DLLME; (Aii) standard mixture (100 μg/L) after DLLME; (B) lake water sample spiked with PAHs (10 μg/L); and (C) wastewater sample. 1, Ace; 2, Flu; 3, Ant; 4, Phe; 5, Flt; 6, Pyr; 7, BaA; 8, BaP." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_21_31418_31431_Figure_1_Chromatograms_of_Ai_standard_mixture_265d7e1014.jpeg 234w," sizes="100vw" width="2100" height="1402"></p><h5><strong>3.1. Evaluation of the Type of Extractants and Dispersants for DLLME</strong></h5><p>Extraction performance in DLLME is strongly influenced by the choice of extraction solvent (i.e., that which is sedimented), which controls the solutes’ partitioning behavior between the aqueous and organic phases. <a href="javascript:void(0);">(24)</a> Five extraction solvents of different polarity, namely, dichloromethane (DCM, dielectric constant (ε): 8.93 <a href="javascript:void(0);">(53)</a>), chloroform (ε: 4.81 <a href="javascript:void(0);">(54)</a>), carbon tetrachloride (ε: 2.24 <a href="javascript:void(0);">(55)</a>), chlorobenzene (ε: 5.62 <a href="javascript:void(0);">(56)</a>), and cyclohexane (ε: 2.02 <a href="javascript:void(0);">(57)</a>), were evaluated. Based on the overall response (i.e., peak areas), dichloromethane outperformed the other chlorinated solvents (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.6c01489#fig2">Figure 2</a>). This might be attributed to the stronger partitioning of PAHs into DCM, which exhibited the highest polarity among the tested extractants. <a href="javascript:void(0);">(58)</a> Therefore, DCM was selected as the extraction solvent. It is known that the dispersive solvent serves as a “bridge” between the aqueous sample and the water-immiscible extractant, and its selection is therefore crucial to DLLME performance. Five dispersive solvents (methanol, ε: 33; <a href="javascript:void(0);">(59)</a> ethanol, ε: 24.5; <a href="javascript:void(0);">(60)</a> isopropanol, ε: 20.1; <a href="javascript:void(0);">(61)</a> acetone, ε: 20.7; <a href="javascript:void(0);">(62)</a> acetonitrile, ε: 36 <a href="javascript:void(0);">(63)</a>) of different polarities were studied. Among these, ACN having the highest polarity provided the highest extraction response. This might be attributed to the reduced interfacial tension, which promotes formation of smaller microdroplets, and increases interfacial area for partitioning process, thereby improving extraction efficiency. <a href="javascript:void(0);">(64)</a> Thus, DCM and ACN were selected as the extractant and dispersant, respectively, for subsequent FrFD and BBD experiments.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_21_31418_31431_Figure_2_Effect_of_A_selection_of_extraction_solvent_B_selection_of_dispersive_solvent_5abd279b17.jpeg" alt="ACS Omega 2026, 11, 21, 31418–31431: Figure 2. Effect of (A) selection of extraction solvent; (B) selection of dispersive solvent." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_21_31418_31431_Figure_2_Effect_of_A_selection_of_extraction_solvent_B_selection_of_dispersive_solvent_5abd279b17.jpeg 135w," sizes="100vw" width="1000" height="1154"></p><h4><strong>4. Conclusion</strong></h4><p>An improved and environmentally friendly DLLME-GC-FID method was described for the quantitative analysis of PAHs in surface water. RSM was applied by first screening influential variables using a fractional factorial design, followed by the Box-Behnken design optimization of the DLLME parameters. In contrast to single-factor experimentation, the fitted model and 3D response surface plots showed factor interactions and their combined effects on extraction performance, facilitating near-optimal conditions to be identified with a limited number of experiments. Using the optimized conditions, low detection limits, good enrichment factors, acceptable reproducibility, and recoveries were achieved. The validated analytical features indicate that the proposed method is robust and cost-effective for the routine screening of PAHs in terrestrial water bodies.</p></div>

Experimental Design-Based Dispersive Liquid–Liquid Microextraction with GC-FID for Determination of Polycyclic Aromatic Hydrocarbons in Surface Water

<p><strong>This study investigates the antioxidant and anti-inflammatory effects of (−)-epicatechin in an in vitro model of metabolic dysfunction-associated steatotic liver disease (MASLD). Lipid peroxidation, inflammatory, and profibrotic markers were assessed alongside ATR-FTIR spectroscopy to evaluate whether spectral changes reflect disease progression and treatment response.</strong></p><p>Epicatechin reduced lipid peroxidation products, TNF-α, and TGF-β1 in a concentration-dependent manner, with stronger protective effects observed during pretreatment. ATR-FTIR spectral changes closely correlated with biochemical markers, demonstrating its potential as a rapid, non-destructive tool for monitoring oxidative stress, inflammation, and fibrosis in MASLD research.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978"><strong>ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD</strong></a></h4><p>Marija Hefer, Nikolina Filipović, Stjepan Šarić, Nela Malatesti, Ana Petrović, Elvira Kovač-Andrić*, and Martina Smolić*</p><p>ACS Omega 2026, 11, 23, 33333–33344</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1021/acsomega.5c09978">https://doi.org/10.1021/acsomega.5c09978</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><p>Metabolic dysfunction-associated steatotic liver disease (MASLD) represents one of the most prevalent chronic liver disorders. Due to its predisposition to progress toward metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC), MASLD has become a significant global health burden and a major challenge for healthcare systems. <a href="javascript:void(0);">(1−4)</a></p><p>The majority of patients with MASLD or MASH are obese and frequently present with additional metabolic comorbidities, most commonly insulin resistance, type 2 diabetes mellitus, and dyslipidemia. <a href="javascript:void(0);">(5−7)</a> Furthermore, most conditions associated with MASLD and MASH are characterized by lipid metabolism impairment, with elevated levels of oxidative stress. Impaired lipid metabolism, together with prolonged oxidative stress, promotes hepatocellular lipotoxicity and mitochondrial dysfunction. These alterations affect the antioxidant defense system and increase reactive oxygen species (ROS) generation, thereby contributing to the onset of MASLD and its progression to MASH, along with more advanced liver diseases. <a href="javascript:void(0);">(8,9)</a></p><p>One of the main results of oxidative stress is lipid peroxidation, in which ROS attack (poly)unsaturated fatty acids within membrane lipids. This initiates a series of structural and functional alterations, where the formation of conjugated dienes (CDs) reflects early double-bond rearrangements in fatty acyl chains, while lipid hydroperoxides (LOOHs) represent key propagation-phase products that destabilize cell membranes and increase oxidative injury. <a href="javascript:void(0);">(10,11)</a> Therefore, determining LOOH and CD levels, along with their overall LOOH/CD ratio, might be useful for determining the lipid peroxidation dynamics and the progression of MASLD.</p><p>Along with oxidative damage to the cell structures, inflammation is also a key regulator of MASLD progression. In this context, tumor necrosis factor-α (TNF-α) is suspected to play a central role as its levels are found to increase alongside MASLD to MASH progression. <a href="javascript:void(0);">(12−14)</a> It has also been found that ROS and TNF-α are closely interconnected, where elevated TNF-α levels could serve as an indicator of oxidative cellular damage and enhanced lipid peroxidation. <a href="javascript:void(0);">(15−17)</a> Often, one of the results of MASLD progression is the development of liver fibrosis, which is usually mediated by transforming growth factor beta 1 (TGF-β<sub>1</sub>) as its levels are frequently elevated in MASH and contribute to the worsening of hepatic steatosis. <a href="javascript:void(0);">(18)</a> <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig1">Figure 1</a> summarizes the pathways associated with MASLD to MASH progression, with emphasis on lipid peroxidation (CDs, LOOHs) and inflammatory/fibrogenic signaling (TNF-α, TGF-β<sub>1</sub>).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_1_Schematic_representation_of_lipid_peroxidation_and_its_role_in_MASLD_progression_e6a8e05d3e.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 1. Schematic representation of lipid peroxidation and its role in MASLD progression. Lipid accumulation in MASLD leads to the release of free radicals and ROS, which attack (poly)unsaturated fatty acids within membrane lipids, leading to lipid peroxidation, inflammation (via TNF-α), and fibrotic changes (via TGF-β1), thereby contributing to the transition from MASLD to MASH and further progression to cirrhosis and HCC. In lipid peroxidation, reactive radicals (R·) subtract the hydrogen from the (poly)unsaturated lipid, thereby resulting in the formation of a lipid radical, which undergoes rearrangements to form a CD. The lipid radical then reacts with molecular oxygen to form an unstable lipid peroxyl radical, which further subtracts a hydrogen from another (poly)unsaturated lipid, thereby resulting in the formation of a LOOH. The subtraction of hydrogen from the (poly)unsaturated lipid also results in the formation of a lipid radical, thereby perpetuating the chain reaction. The figure was created with Servier Medical Art (https://smart.servier.com/) and MarvinSketch (ChemAxon, https://chemaxon.com/marvin)." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_1_Schematic_representation_of_lipid_peroxidation_and_its_role_in_MASLD_progression_e6a8e05d3e.jpeg 216w," sizes="100vw" width="1834" height="1327"></p><p>However, the exact role of different lipid peroxidation indicators and their contribution to MASLD progression toward MASH have not yet been fully elucidated, with the number of studies addressing this issue remaining limited. Given these limitations in research, attention has increasingly turned toward dietary antioxidants, particularly polyphenols or mixtures of polyphenol-rich plant extracts, as potential modulators of oxidative stress and lipid peroxidation in hepatic steatosis. <a href="javascript:void(0);">(19−23)</a></p><p>Among available polyphenols, we selected (−)-epicatechin (EC) because of its strong antioxidant and radical scavenging capacity, with its documented effects on inflammatory signaling. <a href="javascript:void(0);">(24−26)</a> One study even found that the antifibrotic properties of EC were comparable to those of silymarin, with EC showing even stronger reducing effects on malondialdehyde (MDA) formation in vivo. <a href="javascript:void(0);">(27)</a> Since MDA is a secondary product of LOOH breakdown, <a href="javascript:void(0);">(28)</a> these results could link EC’s potential antifibrotic properties to its ability to attenuate lipid peroxidation, thereby potentially mitigating the progression of MASLD to MASH. Unlike more extensively studied compounds such as epigallocatechin gallate, <a href="javascript:void(0);">(29,30)</a> EC has been less investigated in the context of MASLD, providing an opportunity to explore a novel candidate. At the same time, the growing use of advanced spectroscopic techniques offers new opportunities to monitor biochemical alterations in cells and tissues.</p><p>Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy recently emerged as a promising tool for monitoring such changes. The application of ATR-FTIR spectroscopy and similar techniques for steatosis and lipid quantification, along with determining the lipid peroxidation extent, has been introduced, especially in the context of hepatic steatosis. Determining the intensity of C═O vibrational stretching at ∼1740 cm<sup>–1</sup> (triacylglycerols, cholesteryl esters, lipid oxides), as this type of bond usually occurs in lipid peroxidation, could also be valuable for determining the overall extent of steatosis. <a href="javascript:void(0);">(31−33)</a> Along with lipid peroxidation extent, determining the degree of unsaturation in MASLD models is crucial for understanding the changes in cellular components as the degree of unsaturation is often found to decrease due to steatotic changes in cells and tissues. (Poly)unsaturated fatty acids contribute to membrane fluidity and are essential for maintaining membrane integrity and signaling, while their depletion leads to increased membrane rigidity and impaired cellular function. <a href="javascript:void(0);">(34,35)</a> Given their high susceptibility to peroxidation, a reduction in their levels could indicate either an adaptive response to oxidative stress or a disruption of lipid homeostasis. Therefore, correlating the degree of unsaturation (∼3010 cm<sup>–1</sup> for ═C–H) and the intensity of carbonyl stretching (∼1740 cm<sup>–1</sup> for C═O) with the LOOH/CD ratio and cytokines, TNF-α and TGF-β<sub>1</sub>, could be valuable for the noninvasive monitoring of disease progression.</p><h4><strong>Methods</strong></h4><h5><strong>Experimental Design</strong></h5><p>Two experimental models were introduced for the experiments: EC pretreatment and EC Posttreatment models. In both models, the untreated cells (UT) group was used as a negative (vehicle) control group, while the sodium oleate-treated group (OA) was used as a positive control group, with EC (control) being the highest concentration of EC used as a control for the tested compound. In the Pretreatment model, the cells were pretreated with 10, 30, and 50 μM EC for 4 h, followed by a 24 h posttreatment with 1.5 mM sodium oleate. In the Posttreatment model, the cells were treated with 1.5 mM sodium oleate for 12 h, followed by the addition of the same concentrations of EC in the presence of 0.75 mM sodium oleate. All of the experimental groups were labeled as EC50+OA, EC30+OA, and EC10+OA, according to the concentration used in the experiments. EC concentrations of 10, 30, and 50 μM were chosen based on our previous MTS assay and Oil Red O staining results, where we tested 10–100 μM EC to evaluate its effect on MASLD in the Pretreatment model. <a href="javascript:void(0);">(42)</a> As it was proven effective in the Pretreatment model, the Posttreatment model was added to evaluate whether EC could also mitigate oxidative and inflammatory/fibrotic changes once steatosis was already established. Another important consideration was to assess the potential cytotoxicity of the concentrations used, as EC and other polyphenols are often consumed as dietary supplements with poorly regulated dosages. As these cytotoxic effects are often subtle and overlooked in conventional biochemical assays, combining FTIR spectral analysis with complementary assays could provide a more comprehensive assessment of potential cytotoxicity.</p><h5><strong>ATR-FTIR Assessment of Lipid Peroxidation</strong></h5><p>ATR-FTIR spectroscopy was used to determine the extent of lipid peroxidation and steatosis. The lipid samples were dissolved in chloroform and normalized to a lipid concentration of 20 mg/mL. After the evaporation of the solvent, the ATR-FTIR spectra were recorded using an <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=Cary+630"><strong>Agilent Cary 630 FTIR spectrophotometer</strong></a> (Agilent Technologies, Santa Clara, CA, USA), at the Faculty of Biotechnology and Drug Development at the University of Rijeka. All ATR-FTIR spectra were processed using the automatic least-squares baseline correction algorithm implemented in <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library?mainauthorItems=10&amp;search=MicroLab+Expert"><strong>Agilent MicroLab Expert v.10.5 software</strong></a> (4000–650 cm<sup>–1</sup>; clean scans: 4; threshold: 0.002; background scans: 2; sample scans: 100; resolution: 16 cm<sup>–1</sup>; apodization: HappGenzel; phase correct: Mertz; method gain: 221). Spectra of pure chloroform were recorded under identical conditions and subtracted from sample spectra to remove any residual solvent contributions. Chloroform was recorded to be excluded in the background, and the data were collected in the spectral range 3200–1600 cm<sup>–1</sup> on an ATR device. To ensure comparability between samples, the spectra were normalized against the asymmetric CH<sub>2</sub> stretching vibration of lipid acyl chains (2925–2922 cm<sup>–1</sup>), which was used as an internal reference band due to its relative stability across treatments and minimal alteration during lipid peroxidation. Baseline correction was applied prior to normalization to minimize background variability. Relative intensities of lipid peroxidation-associated bands (C═O stretching at 1744–1735 cm<sup>–1</sup> and ═C–H stretching at 3010 cm<sup>–1</sup>) were then expressed as a ratio to the CH<sub>2</sub> reference peak, allowing quantitative comparison of the lipid peroxidation extent between experimental groups. The data were presented as percent relative intensities (%<i>I</i><sub>rel</sub>).</p><h4><strong>Results and Discussion</strong></h4><h5><strong>ATR-FTIR Spectroscopy for the Assessment of Lipid Peroxidation and Steatosis Extent</strong></h5><p>To evaluate the overall steatosis and lipid peroxidation extent in HepG2 cells, ATR-FTIR spectroscopy was used. <a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig5">Figure 5</a> shows the 3200–1600 cm<sup>–1</sup> spectral region, including the key C═O (1744–1735 cm<sup>–1</sup>) and ═C–H (3010 cm<sup>–1</sup>) stretching bands, which correlated with the biochemical parameters measured in this study. The increased intensity of the C═O band in sodium oleate-treated groups corresponds to the accumulation of oxidized lipid species derived from peroxidation of (poly)unsaturated fatty acids. On the other hand, a decrease in the ═C–H stretching band near 3010 cm<sup>–1</sup> indicates a progressive depletion of double bonds which undergo rearrangement to form CDs or further transformations associated with lipid peroxidation.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_5_ATR_FTIR_spectra_of_control_groups_aabfabbdc1.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 5. ATR-FTIR spectra of control groups: UT (blue) and EC (control) (purple); along with experimental groups: OA (yellow), EC50+OA (red), EC30+OA (orange), and EC10+OA (brown) in the wavenumber range 3200–1600 cm–1 for the (a) EC Pretreatment Model and (b) EC Posttreatment Model." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_5_ATR_FTIR_spectra_of_control_groups_aabfabbdc1.jpeg 193w," sizes="100vw" width="1572" height="1272"></p><p>All experimental groups show similar FTIR profiles in the lipid regions; however, to determine the overall extent of lipid peroxidation and steatosis, the relative intensities of ═C–H stretching of unsaturated lipids (3010 cm<sup>–1</sup>) and C═O stretching of esters (1744–1735 cm<sup>–1</sup>) were measured according to the baseline and normalized according to asymmetric CH<sub>2</sub> stretching of lipid chains (2925–2922 cm<sup>–1</sup>) (<a target="_blank" rel="noopener noreferrer" href="https://pubs.acs.org/doi/10.1021/acsomega.5c09978#fig6">Figure 6</a>). This approach was chosen as the greatest changes were noticed among the given intensities of the peaks in all groups.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/ACS_Omega_2026_11_23_33333_33344_Figure_6_Percent_relative_intensities_e921d53b57.jpeg" alt="ACS Omega 2026, 11, 23, 33333–33344: Figure 6. Percent relative intensities (%Irel) of ═C–H stretching (unsaturated lipids) and C═O stretching of esters for the (a) EC Pretreatment Model and (b) EC Posttreatment Model. The results are presented as mean values of %Irel ± SD." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_ACS_Omega_2026_11_23_33333_33344_Figure_6_Percent_relative_intensities_e921d53b57.jpeg 149w," sizes="100vw" width="1000" height="1044"></p><p>Also, as indicated in the literature, the increase in intensities of C═O stretching and a decrease in intensities of ═C–H stretching are mostly associated with an increase in the lipid peroxidation extent. <a href="javascript:void(0);">(33,50)</a> Furthermore, a decrease in ═C–H stretching corresponds to a decrease in the (poly)unsaturated fatty acid content, which is often noted in MASLD/MASH models. <a href="javascript:void(0);">(51,52)</a></p><h4><strong>Conclusion</strong></h4><p>In a sodium oleate-induced hepatic steatosis model, EC showed a concentration-dependent effect on the overall radical scavenging ability of HepG2 cells. However, when EC was given as a posttreatment, these results were not achievable. EC also reduced propagation-phase lipid peroxidation by reducing relative LOOH levels in both models, lowering the LOOH/CD ratio (except for the EC50+OA pretreatment group), and effectively decreasing sodium oleate-induced increases in TNF-α and TGF-β<sub>1</sub> concentrations. ATR-FTIR results were consistent with changes in lipid (per)oxidation and reflected the overall antioxidant and anti-inflammatory effects of EC observed through biochemical assays. Compared with biochemical assays, ATR-FTIR spectroscopy has several advantages, including rapid and nondestructive analysis that requires minimal sample preparation. Considering that it can simultaneously capture multiple molecular changes, this represents a reliable approach for accurate and complementary insights into lipid peroxidation and redox alterations in MASLD. However, it is important to correlate ATR-FTIR spectral changes with biochemical assays to further strengthen its interpretation.</p></div>

ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD

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ATR-FTIR Assessment of the Antioxidant and Anti-Inflammatory Effects of (−)-Epicatechin in MASLD

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