Analytica

The paper describes the preparation of silver-containing nanocomposites by thermolysis of silver cinnamate and their application for the manufacture of reactive indicator paper (RIP) sensitive to iodine. The composition, structure, and properties of the obtained materials were studied using IR spectroscopy, X-ray diffraction, the gravimetric analysis method, scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. Optimal conditions for modifying a cellulose carrier with nanocomposites in laboratory conditions were selected, ensuring high sensitivity of RIP to iodine and uniform and reproducible distribution of the reagent. A new gas extraction colorimetric technique for determining iodide ions in the range of 0.03–1.6 mg L⁻¹ (limit of detection 0.01 mg L⁻¹) was developed, allowing iodides to be determined in multicomponent objects such as food products, pharmaceuticals, and various water bodies with minimized sample preparation. The use of iron(III) as an oxidizing agent and the use of dynamic gas extraction ensure high selectivity and good analytical performance. Full article

Despite its widespread use in pharmaceuticals, cosmetics, and animal nutrition, the environmental implications of nicotinamide remain largely underexplored. Given its high solubility and prevalent presence in wastewaters, nicotinamide has been proposed as a potential environmental marker and emerging contaminant. This study focuses on the evaluation and modeling of medium and ionic strength effects on the acid-base behavior of nicotinamide in aqueous solution. To this aim, the protonation constants of nicotinamide were determined in aqueous media containing various supporting electrolytes (sodium chloride, tetramethylammonium chloride, tetraethylammonium iodide) at T = 298.15 K and different ionic strengths. The medium and ionic strength dependencies were described through well-established thermodynamic models, including the Extended Debye–Hückel equation, the Specific ion Interaction Theory, and the Pitzer approach. Additionally, the Setschenow constant of nicotinamide was determined through distribution measurements between 1-octanol and NaCl_(aq) solutions. Furthermore, a critical data analysis was performed to assess the reliability of the proposed models and provided interaction parameters. Overall, this work thoroughly describes the acid-base behavior of nicotinamide in different aqueous media and its distribution between aqueous and organic phases, offering fundamental insights for the assessment of its chemical speciation in real systems, which is crucial for future applications in environmental monitoring and remediation strategies. Full article

Comparative interpretation of antioxidant activity across complex natural-product extracts and multi-component formulations is often limited by heterogeneous endpoints, assay-specific reporting, and incompatible units. In this study, we establish a dimensionless Effect Factor (EF) framework as a standardized comparative indexing system for matched-condition comparison of antioxidant-related readouts. EF maps four widely used antioxidant-related readouts—ABTS, DPPH, total phenolic content (TPC), and total flavonoid content (TFC)—into a common dimensionless index space while preserving endpoint meaning through two complementary tracks: potency-type EF and content-type EF. Using a standardized extraction and assay pipeline, we generated EF indices for 586 extracts prepared under water and 30% ethanol conditions and organized them into EF-indexed databases. EF is not intended as a predictive or biologically validated efficacy metric, but as a standardized comparative indexing framework that enables reproducible benchmarking, database-level referencing, and structured interpretation of heterogeneous antioxidant-related data under harmonized experimental conditions. These results establish the EF-indexed resource as a scalable and expandable comparative infrastructure for natural-product research. Full article

Arachidonate 5-lipoxygenase (ALOX5) is a key enzyme implicated in several inflammatory disorders, including asthma and allergic rhinitis. Despite its therapeutic importance, only one compound is currently approved as an ALOX5 inhibitor in the United States, highlighting the urgent need for new drug candidates. Progress in this area is often hindered by conventional bioassays, which can be labor-intensive, costly, and unsuitable for complex mixtures. To overcome these challenges, we developed a simple thin-layer chromatography (TLC) bioautographic assay for the rapid detection of ALOX5 inhibitors in natural extracts, a rich source of pharmacologically active compounds. The method exploits the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with 3-(dimethylamino)benzoic acid (DMAB) during the ALOX5-catalyzed conversion of arachidonic acid, producing a colored indamine dye. Experimental parameters influencing chromogenic reaction were investigated and optimized to minimize reagent consumption while ensuring accuracy and sensitivity of the method. The assay was then applied to a panel of natural products and to crude mushroom extracts, enabling the rapid identification of several active compounds within complex extracts, including the dual COX2/ALOX5 inhibitor 3α-acetylpolyporenic acid A. Easy to implement, cost-efficient, and well suited for screening and bioguided fractionation, this TLC bioassay provides a powerful tool to accelerate the discovery of novel anti-inflammatory compounds. Full article

This paper describes a simple and sensitive method for determining the content of five bisphenols at the µg L⁻¹ level in tea infusion samples. The procedure uses a polypropylene hollow fiber filled with a deep eutectic solvent as the acceptor phase and 1-octanol as the supported liquid membrane, and the optimal conditions for the three-phase stir bar microextraction method were established as follows: a menthol–octanoic acid mixture (1:1 molar ratio) as the hollow-fiber filling, an extraction time of 1 h, and 80 µL of methanol for analyte desorption. The method demonstrated good linearity over the ranges of 1.5–30.0 µg L⁻¹ (BPF, BPA, BPAF, and BADGE) and 6.0–120.0 µg L⁻¹ (BPZ), with limits of detection between 0.28 and 1.01 µg L⁻¹, and the relative recovery values were satisfactory (99–120%) with acceptable precision (RSD < 17%). Thus, the method was successfully applied to quantitatively analyze twenty commercial tea infusions, in which BPF was detected at concentrations above the LOQ, and the greenness and overall applicability of the approach were confirmed using the AGREEprep and BAGI assessment tools. Full article

We studied experimentally and computationally the structures and optical properties of sulfur (S), selenium (Se) and tellurium (Te) ring clusters. We encapsulated S, Se and Te into AFI, MOR, CHA and LTA zeolites via vapor adsorption or high-pressure injection from melt and studied Raman and optical absorption spectra (RS and OAS, respectively) of zeolite single crystals with incorporated S, Se and Te ring clusters. Importantly, strict orientation of the rings in zeolite crystals allowed us to study the polarization/orientation dependency of ring RS and OAS. The obtained experimental spectra are found to be in agreement with density functional theory results (DFT using the PBE0 functional and def2-TZVP basis sets) for S₈, Se₆, Se₈, Se₁₂, Te₆ and Te₈ ring molecules. The agreement is especially good for Te rings, while for S and Se rings harmonic frequency scaling factors are required. The S and Se rings display light-induced effects, which we attribute to the presence of conical intersections between their ground and excited electronic states, resulting in isomerization and subsequent fragmentation. We consider this effect using the Se₆ ring example. This phenomenon is important for understanding photostructural changes not only in chalcogen clusters but also in bulk materials such as amorphous selenium. Full article

The species Psychotria densicostata Müll.Arg. is a shrub belonging to the Rubiaceae family, endemic to Brazil. So far, there are reports neither of phytochemical work on nor of biological evaluation of it. This study investigated its alkaloid profile and evaluated the inhibitory effects of extracts, alkaloid-enriched fractions and one of its major constituents on human neutrophil elastase (HNE). The monoterpene indole alkaloids (MIAs) strictosidine (1), (3α,5α)-5-carboxystrictosidine (2), strictosidine lactam (3), lyaloside (4), lyalosidic acid (5), 5-carboxystrictosamide (6), 3,4-dehydrostrictosidinic acid (7), and N-glucopyranosyl vincosamide (8) were characterized in mixture, in its leaves, and/or stems by using an integrated approach combining nuclear magnetic resonance (NMR) techniques, high performance liquid chromatography coupled to a tandem mass spectrometer with an electrospray ionization source (HPLC-ESI-MS/MS), and molecular networks. The crude leaf extract and an alkaloid-enriched fraction derived from it showed inhibitory activity against HNE. These results contribute to the chemical knowledge of the species and suggest its potential biological property. Full article

The herbal tea industry has experienced substantial growth, particularly regarding green tea (Camellia sinensis). In the manufacturing of filter tea, fine herbal dust is generated as a residual by-product during grinding and sieving and is typically discarded as waste. This study aims to explore the application of ultrasound-assisted extraction (UAE) for secondary valorisation of green tea herbal dust by investigating the effects of various parameters on extraction efficiency. Antiradical activity of UAE extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and the total phenolic content (TPC) was measured using Folin–Ciocalteu’s assay. Furthermore, selected phenolics were quantified by HPLC and qualitatively characterised by liquid chromatography coupled with electrospray ionisation and quadrupole time-of-flight tandem mass spectrometry (LC-ESI-QToF-MS/MS). The results demonstrate that UAE parameters have a pronounced influence on the antioxidant activity, TPC, and individual polyphenolic profile of green tea herbal dust extracts. Ethanol–water mixtures at a ratio of around 40–60%, as well as moderate impulse regimes (around 60%) and extraction times (around 10 min), were the most suitable for extracting green tea polyphenols. Epigallocatechin gallate was the predominant phenolic component in most extracts, alongside epicatechin, epigallocatechin, catechin, and gallic acid. The findings highlight the UAE technique as a robust, green, and scalable method for valorising green tea by-products, thereby facilitating the development of high-value natural extracts for applications in the food, pharmaceutical, and cosmetic industries. Full article

C₁₃-Norisoprenoids are important contributors to the aroma of Riesling wine. Their quantification is analytically challenging due to their low concentrations, the lack of commercial standards and their pronounced sensitivity to analytical conditions, reflecting their chemical lability, as well as the dynamic nature of the wine matrix, leading to high reactivity and, consequently, remarkable structural diversity. Here, we developed an assay for the analysis of C₁₃-norisoprenoids in wine using headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry (HS-SPME–GC-MS/MS). After evaluating different fiber materials, a statistical design of experiments (DoE) approach was employed to systematically optimize key HS-SPME parameters, including incubation, extraction and desorption conditions. Selected reaction monitoring (SRM) transitions were established for all targeted C₁₃-norisoprenoids, allowing the assay to provide relative quantification of more than 40 compounds using representative labeled and unlabeled standards to generate linear calibration curves. Following method validation, this approach was applied to a young German Riesling wine to investigate the effect of various acidic hydrolysis conditions on the norisoprenoid profile as well as on specific compounds. A central composite design (CCD) was used to systematically study the impact of pH, temperature, and hydrolysis time. Quantitative data were obtained for 22 C₁₃-norisoprenoids demonstrating that hydrolysis conditions strongly affected the norisoprenoid composition. pH and temperature showed a greater influence than reaction time. Response surface models (RSM) indicated that TDN, Vitispirane and TPB in particular are predominantly formed under strongly acidic and high-temperature conditions, whereas others such as Riesling acetal and actinidols are formed under milder conditions. The results indicate that hydrolysis conditions should be tailored to the specific norisoprenoid under investigation and the research question, particularly when simulating conditions of accelerated wine ageing for analytical purposes. Full article

A quartz crystal microbalance-based biosensor for the specific detection of the first transgenic common bean (L.) cultivar (BRS FC401 RMD) with resistance to Bean golden mosaic virus (BGMV) was developed. The immobilization chemistry relies on the strong bond between the thiolated probe and the gold electrode surface. The probe sequence is internal to a region of the BGMV rep gene that was introduced into the common bean genome. The sensor’s analytical performance was determined using synthetic oligonucleotides. Real samples of transgenic and wild-type bean seeds were also tested. Sample pretreatment consisted only of enzymatic fragmentation, followed by a thermal denaturation step combined with blocking oligonucleotides. Different biosensor regeneration approaches were studied. Immobilization showed good reproducibility (CV% of 5.8%). The biosensor proved specific for both synthetic oligonucleotides and non-amplified genomic DNA. A linear detection range of 0–1.4 ng/µL was observed, with a detection limit of 0.18 ng/µL. Three sequential detections were performed without loss of surface activity. The results demonstrate the biosensor’s potential for direct, real-time, label-free detection of DNA samples for field screening of transgenic common bean cultivars. Full article

Secondary electrospray ionization mass spectrometry (SESI-MS) has emerged as a powerful technique for the real-time, non-invasive analysis of volatile organic compounds (VOCs) in complex matrices, such as exhaled breath and microbial volatilomes. However, its transition to routine application is hindered by significant challenges in absolute quantification, unambiguous identification, and standardization. This review provides a comprehensive overview of these limitations and the emerging solutions proposed to overcome them. Matrix effects, including gas-phase ion suppression and C-trap competition, are examined alongside mitigation strategies such as spectral stitching and standard addition. To enhance quantification stability, advanced standard delivery systems and dynamic quality control protocols are evaluated. The identification bottleneck—stemming from the absence of chromatographic separation—is addressed through the use of curated databases and advanced fragmentation techniques, such as incremental quadrupole acquisition to resolve overlapping spectra (IQAROS), to resolve isobaric interferences. Furthermore, the role of chemometrics in extracting biological fingerprints is discussed. Finally, the need for harmonized reporting standards and multicenter validation is emphasized to ensure cross-study reproducibility. Resolving these methodological gaps is essential for the clinical and industrial translation of SESI-MS. Full article

Natural deep eutectic solvents (NADES) offer sustainable alternatives to conventional solvents for plant extraction, yet their influence on extract composition and bioactivity preservation requires further study. Here, choline chloride-based NADES with lactic acid or propylene glycol were evaluated for ultrasound-assisted extraction (60 °C, 30 min, 1:20 w/v) of polyphenol-rich fractions from Sanguisorba officinalis and Symphytum officinale. Spectrophotometric analysis yielded total phenolic contents of 6.49–9.67 mg GAE g⁻¹ and total flavonoids of 0.08–0.52 mg g⁻¹, with values dependent on the plant matrix and the NADES formulation. Targeted HPLC-MS/MS enabled identification of representative phenolic acids (chlorogenic, caffeic, ferulic, rosmarinic) and flavonoid markers (rutin, quercetin derivatives), showing qualitative differences in the detected marker profiles between solvents and matrices. Functional assays demonstrated pronounced antioxidant-related effects, including DPPH radical scavenging at 0.5–25 µg mL⁻¹ (polyphenols), inhibition of lipid peroxidation in rat erythrocytes at 0.25–1.20 µg mL⁻¹, and modulation of mitochondrial respiration and permeability transition in isolated rat liver mitochondria. Overall, the results indicate that choline chloride-based NADES can be used to obtain polyphenol-rich plant extracts compatible with the applied analytical workflow while preserving redox-active fractions, supporting their utility in green analytical sample preparation. Full article

Phosphonyl tyrosine is one of the main biomarkers to confirm exposure to highly lethal organophosphorus nerve agents (OPNAs) in vivo. However, a critical challenge remains unresolved: ionization suppression occurs during the analysis of phosphonyl tyrosine by high-resolution mass spectrometry (HRMS) or tandem mass spectrometry (MS/MS), which is induced by the high concentrations of free amino acids present in the digestion solution. In this study, based on the broad-spectrum immunomagnetic beads with high affinity antibodies, a non-targeted early warning and verification strategy was developed. Compared with the recommended operating procedures for analysis in the verification of chemical disarmament, the total analysis time was reduced from several hours to about 30 min. Moreover, the detection sensitivity was increased by nearly one order of magnitude, and the detection limit (LOD) was 0.01 ng/mL. Furthermore, the screening strategy can cover all OPNAs listed as 1A.01, 1A.02 and 1A.03 in Schedule 1 of the CWC. Therefore, we have developed a rapid, sensitive, and broad-spectrum approach to accurately screen for OPNAs exposure, while also offering a novel strategy and technical support for chemical defense and occupational health assessment. Full article

Impurity profiling is of significant analytical and regulatory importance, particularly in the context of lifecycle quality management. A robust chaotropic chromatography method was developed for the determination of olanzapine and its two oxidative degradation products in tablets, in accordance with the ICH Q14 guideline and the principles of Analytical Quality by Design (AQbD). Risk assessment was performed using a combination of the Ishikawa diagram, CNX (Control, Noise and eXperimental) classification, and Failure Mode and Effect Analysis (FMEA). This multistep evaluation identified the critical analytical procedure parameters (APPs) as the acetonitrile content in the mobile phase, the concentration of perchloric acid in the aqueous phase, and the pH of the aqueous phase. These APPs were studied using an experimental design approach to model their effects on key analytical procedure attributes and to compute a multidimensional design space. Robust optimization supported by Monte Carlo simulations ensured compliance with predefined acceptance criteria with a probability of at least 95%. Method validation demonstrated adequate selectivity, limits of quantification of 0.75 µg/mL and 0.5 µg/mL for impurities B and D, linearity with correlation coefficients ≥0.990, accuracy of 98–102% for olanzapine and 70–130% for impurities, and repeatability with RSD ≤2% for the assay and ≤10% for impurities. The method was successfully applied to commercial tablet analysis. Full article

Light-sensitive protecting chemical groups play an important role in the control of chemical reactions with high spatial and temporal resolution. Various forms of o-nitrobenzyl as a protecting compound are used for light-directed DNA synthesis. The suitability of a particular derivative for the application is defined by its photolytic efficiency, a characteristic, that is commonly extracted from a repetitive HPLC procedure. Here, using an example of a phosphoramidite compound with improved properties of deprotection, we delineate a simplified and economic approach based on a measurement of absorbance spectra to evaluate the photolytic efficiency. The obtained values are in close agreement with those determined previously. Full article

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful technique for polymer identification because it can ionize intact macromolecules with large molecular weights (>100 kDa) and detect them over a theoretically unlimited mass range. However, limited mass resolution, accuracy, and sensitivity in the high-mass region restrict detailed structural characterization of polymers. Depolymerization into smaller fragments using established chemistry, such as transesterification, provides a versatile alternative approach. The resulting characteristic fragments (<3 kDa) are polymer-specific and enable both polymer identification and detailed structural analysis, benefiting from the higher mass accuracy and sensitivity in the low-mass range and allowing MS/MS-based identification. To establish general sample-preparation conditions, this study systematically investigated the effects of transesterification of biodegradable polyesters using MeO⁻/MeOH, EtO⁻/EtOH, and t-BuO⁻/t-BuOH on subsequent MALDI-TOF MS analysis. Among the reagents examined, EtO⁻/EtOH combined with THAP/THF matrix treatment was found to be the most efficient, enabling rapid polyester analysis by MALDI-TOF MS within 10 min when the reaction time and reagent concentration were carefully controlled within an optimal window. Full article

The forensic reliability of biological fluids in sexual assault investigations depends on substrate type, environmental exposure, time since deposition, and analytical methodology. This systematic review evaluates the forensic reliability of major biological fluids, semen, blood, saliva, and vaginal secretions by comparing detectability and persistence on porous and non-porous substrates, assessing environmental and temporal effects on DNA integrity, and examining the performance of identification methods. A systematic search of PubMed, Scopus, and Web of Science (2001–2025) was conducted following PRISMA 2020 guidelines. Eligible studies investigated fluid persistence, degradation, or identification reliability under controlled or casework-relevant conditions. A weighted scoring framework categorised relative reliability. Twenty-seven studies met inclusion criteria. Semen and blood demonstrated higher reliability across substrates, particularly when collected within recommended timeframes. Porous substrates reduced surface detectability but occasionally preserved DNA from rapid degradation. Elevated temperature, humidity, and prolonged intervals consistently reduced DNA quality and detection success. Molecular approaches, including mRNA profiling, showed enhanced specificity in degraded or mixed samples, though methodological variability limited direct comparability across studies. The forensic reliability of biological fluids is context-dependent, shaped by complex interactions between substrate characteristics, environmental exposure, and analytical technique. Semen and blood remain robust DNA sources, while emerging technologies offer improved specificity in challenging scenarios. Standardised evaluation frameworks and timely evidence collection remain essential to enhance evidential value and minimise misinterpretation in sexual assault investigations. Full article

Objectives: Using high-performance liquid chromatography (HPLC), we developed and validated an in vitro assay for the quantitative determination of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) activity, supplementing limited current methodologies to assess the efficacy of BACE1 inhibitor compounds. A hexa-histidine tagged peptide substrate of BACE1 was used as the analyte for the determination of in vitro BACE1 activity; it was validated according to ICH guidelines. Methods: The HPLC analysis was performed on the Agilent 1290 Series Infinity II UHPLC System equipped with a Phenomenex Kinetex EVO C18 (100 × 3 mm) 5 µm column. The method was developed using a gradient programme comprising 10% aqueous acetonitrile (0.02 M TFA) to 30% aqueous acetonitrile (0.02 M TFA) for 5 min at a flow rate of 0.6 mL/min. Results: The method showed linearity over the range of 14.92 to 72 µM with ${\mathrm{r}}^2=0.9997$. The accuracy of the method in terms of mean recovery ranged between 96.62 and 98.38%. The %RSD for intra- and inter-day precision was less than 5%. Two commercial inhibitors, AZD3839 and OM99-2, were used to evaluate the performance of the method at their respective IC₅₀, resulting in inhibition of 53.46 and 50.74%, respectively. The described method addresses the void for a practical and cheap alternative to quantitatively determine the activity of BACE1 compared to current commercially available detection assays. Conclusions: We have successfully developed an HPLC method to measure the inhibitory function of two commercial inhibitors of BACE1, indicating the suitability of the method for the identification and characterisation of novel BACE1 inhibitors. Full article

White Analytical Chemistry (WAC) provides a holistic framework for evaluating analytical methods by balancing analytical performance, environmental sustainability, and practical efficiency. Existing WAC assessment tools offer structured evaluation but often lack flexibility or comprehensiveness. To bridge this gap, we introduce the Whiteness Evaluation for Chemical Analysis (WECA) tool as a dynamic, web-based application that enables customizable, context-aware assessment of analytical methods. WECA allows users to select 2–4 criteria per RGB domain (Red: analytical performance; Green: environmental impact; Blue: practical efficiency), assign user-defined weights, and visualize results through an intuitive color-coded interface. The tool calculates a composite WECA score (%) that reflects overall method “whiteness”. Three case studies, covering HPLC-DAD, micellar electrokinetic chromatography, and electrochemical sensing, demonstrate WECA’s applicability and its ability to highlight method strengths and weaknesses across diverse analytical scenarios. WECA represents a step toward more adaptable, transparent, and visually intuitive method evaluation in alignment with the evolving principles of WAC. Full article