Analytica, Volume 7, Issue 3 (September 2026) – 9 articles

The evaluation of analytical methods has evolved beyond traditional validation parameters to encompass environmental sustainability, analytical reliability, and practical applicability. While numerous assessment tools exist for individual dimensions, integrated multicolor frameworks often impose rigid parameter selection, assign equal weights to all dimensions regardless of analytical context, or lack the granular diagnostic feedback necessary for method improvement. To address these limitations, we present the Triple-Color Assessment (TriCA) tool, a web-based platform that integrates three validated metrics into a unified scoring system with a hierarchical weighting scheme. TriCA combines the Analytical Green Star Area (AGSA) for greenness assessment, the Click Analytical Chemistry Index (CACI) for blueness evaluation, and the Analytical Method Reliability Index (AMRI) for redness scoring. While these metrics are provided as defaults, users may select alternative assessment tools within each dimension according to their specific preferences or requirements. TriCA features a user-customizable weighting system with recommended defaults (red = 3, blue = 2, green = 1), reflecting the logical priority that a method must first be valid, then practical, and finally green. Users can adjust these weights for specific contexts, while the underlying metric scoring protocols (AGSA, CACI, and AMRI) remain fixed to ensure reproducibility. Beyond composite scoring, TriCA generates three detailed pictograms, 12 segments for AGSA, eight segments for CACI, and 10 segments for AMRI, each color-coded to show performance at the individual criterion level. This diagnostic capability enables analysts to identify exactly which green chemistry principles, practicality criteria, or validation parameters are deficient, transforming assessment from an opaque scoring exercise into actionable guidance for method improvement. Seven case studies encompassing diverse analytical techniques demonstrate TriCA’s discriminative power and diagnostic utility. The tool is freely accessible at bit.ly/TriCA, offering the analytical community a transparent, reproducible, and diagnostically rich framework for method evaluation, development, and optimization. Full article

Urinary urea is a key biomarker for tracking protein oxidation and nitrogen balance. A persistent analytical limitation is that conventional total-nitrogen assays do not deconvolute urinary urea from urinary ammonia, and direct measurement still requires capital-intensive clinical analyzers. Here, we implement and independently validate two cost-effective methods that resolve urinary urea by pairing the presence and absence of urease: Method 1 is a urease-coupled potentiometric assay using an ammonium ion-selective electrode (ISE method); Method 2 is a microplate Berthelot–salicylate colorimetric assay employing a repurposed consumer-grade aquarium ammonia test kit (API^®) (API method). Each method was validated against the Roche Cobas C311 clinical analyzer (ISE for urinary ammonia and API for urinary urea) in healthy adult volunteers (n = 10 each), and API–ISE were then compared head-to-head for urinary urea. ISE vs. Cobas yielded R² = 0.967, slope 95% CI [0.867, 1.173], and Bland–Altman bias of −1.02 mM (95% LoA [−7.40, +5.36] mM). API vs. Cobas yielded R² = 0.973, slope 95% CI [0.790, 1.039], and bias of −1.55 mM (95% LoA [−11.56, +8.46] mM). API–ISE urinary urea comparison confirmed mutual agreement (R² = 0.9976, slope 95% CI [0.946, 1.024]; bias of +3.47 mM; 95% LoA [−14.60, +21.53] mM), with mean recoveries near 100% (API: 100.7 ± 2.9%; ISE: 100.6 ± 5.4%; 20–350 mM). Both methods agreed with the clinical analyzer, supporting their potential for decentralized, resource-limited testing; clinical and point-of-care validations are needed. Full article

Metabolomic aging clocks—quantitative models that estimate biological age based on small-molecule profiles in biological fluids—have become dynamic tools for assessing individual aging trajectories and predicting the risk of age-related diseases. Although NMR-based approaches have been previously reviewed, the analytical landscape of mass spectrometry (MS)-based metabolomic clocks has not been systematically assessed. This review examines the key components of the MS-based analytical workflow underlying the development of metabolomic aging clocks, including biological matrix selection, chromatographic separation strategies, MS instrumentation, data preprocessing, metabolite annotation, and machine learning-based modeling approaches. Twelve published studies on MS-based metabolomic clocks were identified and systematically compared. These clocks provide predictions of chronological age with mean absolute errors of 3.5–10 years and demonstrate robust associations between metabolomic age acceleration and cardiometabolic risk, frailty, and mortality. Recurrent age-related metabolite classes include tryptophan–kynurenine pathway metabolites, acylcarnitines, sphingolipids, modified nucleosides, and tricarboxylic acid (TCA) cycle intermediates. Currently, liquid chromatography with electrospray ionization (LC-ESI) coupled to high-resolution QTOF or Orbitrap instruments dominates current workflows, with elastic net regression being the most commonly applied modeling strategy. Significant heterogeneity in analytical conditions, incomplete methodological descriptions, and limited cross-study validation remain key obstacles to clinical application. Standardization of pre-analytical protocols, the use of certified reference materials, and harmonized validation frameworks are identified as critical priorities for advancing mass spectrometry-based metabolomic clocks toward clinical translation. Full article

Sulfated polysaccharides from red algae are multifunctional hydrocolloids whose properties depend strongly on molecular structure, sulfate content, and chain conformation. In this study, a sulfated galactan extracted from Gracilaria vermiculophylla collected from a coastal region of northwestern Mexico was investigated to elucidate its structural and functional properties. The polysaccharide was extracted by enzymatic treatment and characterized by monosaccharide analysis, FTIR, 1D/2D NMR spectroscopy, and size-exclusion chromatography coupled with multi-angle light scattering and viscometry. The extracted polysaccharide exhibited high carbohydrate (97%) and sulfate (28%) contents, with galactose as the predominant monosaccharide. It showed a moderate molecular weight (Mw = 2.39 × 10⁵ g/mol), a relatively narrow molar mass distribution (Mw/Mn = 1.23), a high intrinsic viscosity (420.9 mL/g), and an Rg/Rh ratio of 2.2, consistent with an expanded macromolecular conformation in solution. The sulfated polysaccharide also exhibited measurable antioxidant activity, particularly in the ABTS⁺ assay (IC₅₀ = 31.8 ± 2.5 mg/mL) compared with the DPPH assay (IC₅₀ = 51.1 ± 3.0 mg/mL), and showed Fe³⁺-responsive association, leading to the formation of microgel-like structures. These findings indicate that the sulfated galactan characterized in this study is a structurally complex and functionally active hydrocolloid with potential relevance for functional food systems and related biomaterial applications. Full article

Serum albumin binding plays a critical role in determining the pharmacokinetic and pharmacodynamic behavior of many drugs. Despite extensive studies on human and bovine serum albumin, quantitative binding data for rabbit serum albumin remain limited for several therapeutically important compounds. In this study, the binding interactions of three oxicam-class non-steroidal anti-inflammatory drugs (meloxicam, tenoxicam, and piroxicam) with rabbit serum albumin were investigated using circular dichroism spectroscopy and fluorescence quenching measurements. Binding parameters were estimated using nonlinear mixed-effects models that simultaneously incorporated data from multiple spectral channels and experimental replicates while accounting for residual correlation and heteroscedastic variance. Fluorescence-based analyses produced estimates consistent with the combined models and showed substantially lower uncertainty compared with CD-only analyses. These findings demonstrate that fluorescence quenching provides more reliable quantitative estimates of albumin binding affinity, whereas CD measurements exhibit higher variability in this context. The results represent the first systematic report of oxicams binding to rabbit serum albumin, with stability constants of meloxicam (logK = 4.840 ± 0.009), piroxicam (logK = 5.068 ± 0.007 and tenoxicam (logK = 4.668 ± 0.004) and highlight the importance of experimental determination of binding parameters even within closely related drug classes. The modeling framework employed here provides a robust statistical approach for analyzing spectroscopic titration data and may be broadly applicable to studies of drug–protein interactions. Full article

Synthetic azo dyes may be fraudulently added to paprika powder to intensify color, creating a need for rapid, at-line screening before confirmatory chromatographic analysis. This study evaluated handheld laser-induced breakdown spectroscopy (LIBS) for the screening of sodium-salt azo dye adulteration represented by Allura Red (E129), Ponceau 4R (E124), and Orange II in paprika powder. Paprika was spiked at 17 levels (0–6% w/w) in three independent batches, mixed with Al₂O₃ binder, dried, pelletized, and measured with a portable SciAps Z-903 under argon in the broad 190–950 nm region. The calibration models were developed using SNV-pretreated spectra and partial least squares regression with test-set validation based on a 70/30 split by concentration level. The main concentration-related response was increased Na emission, consistent with the sodium-salt form of the dyes, while Al emission showed an inverse matrix-/plasma-coupled trend despite the constant binder fraction. Full-spectrum models gave R²_TSV values of 0.845–0.875 and RMSETSV values of 0.638–0.716% (w/w), using 4–5 latent variables. Outlier trimming improved prediction for Allura Red and Orange II, while regression coefficient-uncertainty variable selection reduced model complexity to 2–3 latent variables with dye-dependent effects. Since sodium-salt azo dyes are commonly used as adulterants in paprika powder, the Na-dominated LIBS response demonstrates good potential for rapid at-line screening. Nevertheless, as LIBS does not directly confirm dye identity, complementary confirmatory analysis using spectroscopic or chromatographic methods remains necessary. Full article

Candida spp. can cause systemic infections with high mortality in immunocompromised patients. Phytotherapy may be an alternative for adjunctive treatment of fungal infections. This study evaluated the phytochemical profile, cytotoxicity, genotoxicity, and antibiofilm activity of the hydroalcoholic extract of Origanum vulgare L. against Candida albicans, Candida tropicalis, and Candida dubliniensis. The extraction and quantification (flavonoids and phenols) were performed, and its antioxidant activity (DPPH) and the presence of bio-active compounds were investigated using high-performance liquid chromatography with Diode Array Detection (HPLC-DAD). Cytotoxicity and genotoxicity tests were conducted using HaCat cell lines. Antifungal activity on planktonic cultures was evaluated using the standard (CLSI M27-S4). The analysis of the extract on biofilms was verified with different exposure times. The extract demonstrated the presence of bioactive molecules, and antioxidant activity. The cytotoxicity test showed viability >70%. Genotoxicity revealed the presence of a few micronuclei in some dilutions. The Minimum Fungicidal Concentration was obtained for C. albicans and C. tropicalis. In the biofilm analysis, there was a reduction of more than 60% in all Candida spp. species at the 24 h exposure time. The findings suggest that the O. vulgare extract exhibits activity against Candida spp. and shows biocompatibility in human keratinocytes. Full article

This study developed and validated a modified QuEChERS extraction procedure for the determination of 32 pesticide residues in tea (Camellia sinensis) found in Moroccan market originating from China and rosemary (Salvia rosmarinus) leaves using UPLC-MS/MS. A total of 202 pesticide residues were initially screened using the NF EN 15662 method, and 32 compounds showed satisfactory recovery and precision in tea. The optimized method, based on freezing before extraction and using PSA + BCG as purification sorbents, achieved recoveries of 70–120% for 97% of analytes. Calibration linearity ranged from 0.001 to 0.2 mg/kg (R² ≥ 0.980), with spiking levels at 0.01–0.1 mg/kg analyzed in duplicate per level and per matrix. Results of LOQ and LOD are compared to multiple MRL databases of Tea (EU, USA, Canada, Codex Alimentarius) despite the scarcity of tea MRLs in most databases, providing an accurate representation of the complexity of the analysis. Uncertainty was evaluated through both validation-based and Horwitz-based approaches, allowing a robust comparison of theoretical and experimental variability. The optimized method was also applied to rosemary, where 31 of the 32 pesticides validated in tea showed satisfactory performance. Similar recoveries, precision, and LOQs were obtained for both matrices, demonstrating the robustness of the method across chemically diverse herbal substrates. Only carbofuran exhibited reduced performance in rosemary, requiring an increased functional LOQ because of matrix-induced ion suppression. This dual-matrix approach provides a reliable and reproducible method for pesticide monitoring in herbal products. This dual-matrix approach provides a reliable and reproducible method for pesticide monitoring in herbal products. Full article

The utilisation of cosmetic plant waxes as sustainable functional ingredients in personal care formulations is on the rise. This is due to their emollient, structuring and film-forming properties. Nevertheless, the intricate lipid composition of these substances engenders considerable analytical difficulties, impeding the reliable characterisation and quality assessment thereof. In this study, an optimised gas chromatography–mass spectrometry (GC–MS) workflow was developed and evaluated for the compositional profiling of waxes derived from R. succedanea and R. verniciflua, two East Asian botanical species that have historically been utilised in cosmetic and lacquer-related applications. Wax samples originating from China, Vietnam, Korea, and Japan were extracted using solvents of different polarity, including petroleum ether, dichloromethane (DCM), ethyl acetate, and acetone. Following the process of BSTFA derivatisation, the extracts were subjected to analysis by GC–MS to evaluate the efficiency of the extraction process and the chemical composition of the extracts. DCM was found to provide the highest extraction yields and the most representative compositional profiles. Across all samples, palmitic acid was identified as the predominant constituent (80–90%), followed by lower amounts of oleic acid, stearic acid, and minor monoacylglycerols. The proposed analytical workflow exhibited satisfactory reproducibility and effective discrimination of lipid constituents, thereby substantiating its application in comparative compositional evaluation, potential support for future quality assessment studies, and formulation development of cosmetic plant waxes. Full article