Conventional phytochemical preparation from botanical materials generally relies on discontinuous batch operations involving extraction, liquid-liquid partitioning, solvent concentration and preparative chromatography, resulting in limited process continuity and automation. In this study, an integrated continuous online platform was developed by coupling continuous ultrasound-assisted stirring extraction (CUASE), centrifugal phase separation (CE), a continuous concentration system (CCS), and parallel semi-preparative chromatography (PSPC). A dynamic emulsified extraction medium consisting of petroleum ether, ethanol and water (5:1:5, v/v/v) enabled simultaneous extraction and phase partitioning of hydrophilic and lipophilic phytochemicals, while the separated phases were continuously concentrated and directly transferred to parallel chromatographic systems for uninterrupted purification. The operating conditions of the CUASE and CCS were systematically optimized to ensure stable continuous operation. Using green tea as a representative botanical matrix, the integrated platform continuously isolated fifteen phytochemicals, including phenolic acids, catechins, purine alkaloids, chlorophylls and carotenoids, with purities exceeding 98.5%. A total of 11.42 g of purified products was obtained during a 3 h continuous operation. Compared with conventional batch-wise workflows, the proposed platform substantially improves process continuity, automation and preparative efficiency by integrating extraction, phase separation, concentration and preparative chromatography into a unified workflow. This work provides a practical engineering strategy for continuous and scalable preparation of phytochemicals from complex botanical matrices.

Understanding root exudation in natural soils remains a methodological challenge because of the complexity of the rhizosphere and rapid assimilation of exuded compounds by soil microbiota. To overcome this challenge, this study aims to develop a novel analytical strategy combining sampling via the EcoRoot methodology with [13]CO2 pulse labeling (PL) and gas chromatography coupled with Orbitrap high resolution mass spectrometry (GCHRMS). Applied to Linum usitatissimum (flax) under both current and simulated 2050 climate conditions, this approach enabled to unambiguously trap and analyze primary metabolites liberated through root exudation, notably monosaccharides. This study demonstrates that the high resolving power of the Orbitrap (60,000 resolution) is mandatory to accurately separate isobaric isotopologues involving [13]C and derivatization-induced [29]Si and allowed to revealed the incorporation of [13]C into specific sugar fragments. Despite identified space-charge effect at low ion intensities (threshold defined at I(M0/TIC)>0.1), isotopic enrichment was successfully quantified, reaching up to 27% of labeled molecules (%LM) for galactose 5TMS, with a mean isotopic enrichment (MIE) of 0.45 for the m/z 217.10746 fragment. Moreover, climate change simulation led to increased exudation of labeled sugars, with M1 fraction, rising from 5% (control) to 8% (climate change) (p < 0.05). This suggests altered carbon allocation strategies in response to environmental stress. GCHRMS enrichment results were compared with the ones measured by GC-combustion-IRMS and were found to be of the same order of magnitude. This finding validates the relevance of this approach while offering key advantages such as compatibility with untargeted analysis and improved sensitivity for low-abundance compounds.

This study describes the separation of no-carrier-added (n.c.a.) [199]Au from thermal neutron irradiated natural platinum targets using two extraction chromatographic resins: TEVA containing a quaternary amine (Aliquat 336) and TK200 impregnated with trioctylphosphine oxide (TOPO, Cyanex 921). The behavior of Au, Pt, and Ir in HCl media (0.1-10 mol·L[-1] HCl) is examined through batch distribution coefficient (Kd) measurements and dynamic column experiments. For TEVA, the Kd of Au remains high across the entire HCl range (2-3·10[4] mL·g[-1]), whereas Pt shows a maximum Kd of 3.3·10[4] mL·g[-1] at 1 mol·L[-1] HCl, decreasing at higher HCl concentration. Iridium exhibits low Kd values ((10-4.6·10[2] mL·g[-1]), consistent with its presence as trivalent species forming weaker complexes with quaternary amines. For TK200, Au Kd ranges from 2·10[5] to 5·10[5] mL·g[-1], with a slight decrease at high acidity. Pt(IV) reaches a maximum Kd of 2.2·10[4] mL·g[-1] at 1 mol·L[-1] HCl. A notable increase in Ir Kd is observed below 1 mol·L[-1] HCl, attributed to hydrolysis of iridium chloro complexes. Based on these distribution data, column separation is achieved in acidic media using 1 g of chromatographic resins. Gold is eluted using dilute HCl with thiourea. The separation factors (SFPt/Au) are 1.6·10[4] for TEVA and 5.9·10[4] for TK200, while the Pt decontamination factors are 158 and 650, respectively, with overall [199]Au yields of 97.6% and 94.1%. The dynamic Pt capacities at 1 mol·L[-1] HCl are 0.13 mmol·g[-1] for TEVA and 0.22 mmol·g[-1] for TK200. The developed separation schemes provide efficient methods for producing medically relevant n.c.a. [199]Au from irradiated platinum targets.

Dynamic high-performance liquid chromatography (DHPLC) was employed as a primary analytical tool to investigate the configurational lability of a chiral tetrahydrofuran-based hemiaminal (1), obtained via nickel-catalyzed electrochemical C-H amination. On a polysaccharide-based chiral stationary phase containing amylose tris(3,5-dimethylphenylcarbamate) as the chiral selector, using n-hexane-ethanol 60:40 as the mobile phase, compound 1 exhibited on-column interconversion between enantiomeric forms, directly evidenced by temperature- and flow rate-dependent peak deformation and plateau formation phenomena. Systematic variation of chromatographic conditions, including temperature and acidic or basic additives, enabled detailed characterization of interconversion kinetics and identification of the transition between chromatographic and dynamic regimes. At 35 °C, the addition of 0.01% trifluoroacetic acid lowered the barrier from 22.7 kcal mol[-1] to 21.1 kcal mol[-1], while in the presence of 0.1% diethylamine, a value of about 20 kcal mol[-1] was determined at 10 °C. Complementary off-column kinetic measurements and solution-state NMR studies revealed that acid- and base-mediated racemization proceeds through two distinct mechanistic pathways. Under these conditions, additional chromatographic peaks were observed and assigned to species involved in the racemization process. Overall, this study highlights dynamic chromatography as a powerful and versatile approach for probing the stereochemical stability of chiral compounds and for gaining mechanistic insight into their interconversion processes.

Estrogens are used to increase milk production but can accumulate in the human body, eventually causing adverse effects. To extract estrogens from milk, a melamine sponge stir bar adsorbent was fabricated in a dual-polymer functionalization process, using polydopamine (PDA), and polyvinyl alcohol (PVA) combined with MIL-125 (MS-PDA@PVA-MIL-125). PDA introduced additional hydroxyl and amino groups to the adsorbent surface, increasing the number of active sites available to the target analytes. PVA acted as a binder to anchor MIL-125 into the composite structure. MIL-125 was employed as the active adsorption material, significantly enhancing the extraction efficiency of estrogens through hydrogen bonding, hydrophobic interaction, and π-π interaction. The composite adsorbent extracted estradiol, ethinyl estradiol, estrone, and hexestrol from milk samples in a stir bar sorptive extraction (SBSE) procedure. The extracted estrogens were determined by high-performance liquid chromatography coupled with diode-array detector. The developed method demonstrated good linearity from 1.50 to 100 μg L[-1] with a limit of detection of 0.50 μg L[-1]. Recoveries from spiked milk samples were in the range of 80 to 104%, with RSDs less than 6%. This innovative design provides high extraction efficiency, simple preparation, self-stirring capability, cost-effectiveness, and good environmental friendliness.

Ilex asprella is a traditional medicinal and edible plant with multiple pharmacological activities such as anti-inflammatory properties. Previous studies on its chemical constituents and pharmacology have mostly focused on roots and stems, whereas research on the leaves remains relatively limited. In this study, an offline comprehensive two-dimensional liquid chromatography‑electrochemical detection (LC × LC-ECD) method combined with LC-HRMS was established for the rapid screening and structural identification of antioxidant components in Ilex asprella leaves. LC × LC-ECD achieved efficient separation of components with wide polarity ranges and significant concentration differences. Using LC-HRMS, a total of 48 compounds were identified or annotated, primarily including 19 hydroxycinnamoyl quinic acids, 14 flavonol glycosides, 6 dihydroflavone glycosides, 5 simple phenolic acids and their glycosides, 2 coumarins, and 2 other compounds. In vitro assays indicated that all electrochemically active fractions obtained from the first-dimension separation exhibited varying degrees of antioxidant activity, and their ABTS and DPPH radical scavenging activities were positively correlated with the electrochemical detection response intensity (R[2] > 0.95). Subsequently, liquid chromatography-electrochemical detection was utilized to compare the antioxidant components among 29 batches of Ilex asprella leaves from different origins. Multivariate statistical analyses, including hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis, consistently demonstrated that the 29 batches of samples could be clearly grouped into three categories based on their antioxidant component profiles, and the classification results were highly correlated with geographical origins, confirming significant variations in the antioxidant component profiles of Ilex asprella leaves from different origins.

Pharmaceuticals are increasingly found as emerging environmental contaminants in water. Many of these compounds may interact with humic acid and other forms of natural dissolved organic matter, which affects properties such as their bioavailability and persistence. Ultrafast affinity extraction (UAE) has been previously applied to study the binding of drugs to proteins for biomedical applications. In this study, UAE was adapted and used to examine humic acid binding to ciprofloxacin and norfloxacin, two antibiotics that have been found in the environment. Microscale columns containing entrapped humic acid within porous silica were prepared and used to capture the non-bound and free fractions of these drugs in solution-phase mixtures containing soluble Aldrich or Leonardite humic acid. The results were used to acquire both the apparent dissociation rate constants and binding constants for the drugs with these types of humic acid. The distribution equilibrium constants measured for this binding at pH 7.0 in 0.10 M potassium phosphate buffer and at 25 °C were 3.6-5.6 × 10[4] L/kg for Aldrich humic acid with ciprofloxacin or norfloxacin and 7.4-8.3 × 10[4] L/kg for Leonardite humic acid. These binding constants fit well with those determined using other techniques. The apparent dissociation rate constants measured by UAE in the same pH 7.0 buffer and at 25 °C were 0.10-0.17 s[-1] for Aldrich humic acid with ciprofloxacin or norfloxacin and 0.052-0.16 s[-1] for Leonardite humic acid. Advantages of this method included its speed, need for only small amounts of soluble drugs and binding agents, and ability to provide information on both binding strength and dissociation rates for these systems. This approach is a general one that could also be applied for binding and rate studies with other emerging contaminants and soluble binding agents for environmental analysis and research.

A simple, fast, and eco-friendly modified dispersive solid-phase extraction (dSPE) method was proposed for the determination of benzophenones and parabens in water samples using biochars derived from nutshells (walnut, hazelnut, peanut, and pistachio shells). Biosorbents were characterized by FTIR, SEM, and BET analyses. The extraction conditions, including sample pH, sorbent mass, extraction time, desorption solvent, volume of desorption solvent, and number of elution cycles, were optimized to achieve maximum efficiency. The optimized dSPE method showed satisfactory performance with recoveries, carried out by analyzing spiked water samples, ranging from 60 to 103% for benzophenones, and from 48 to 97% for parabens. Furthermore, the enrichment factors were achieved in the range of 120 - 258. The results of this study suggest that nutshell-derived biochars have significant potential in the dSPE method for determining target emerging contaminants in water samples. Additionally, the developed methodology was evaluated in terms of applicability and sustainability using the Blue Applicability Grade Index (BAGI), Analytical Greenness Metric for Samples Preparation (AGREEprep), and Sample Preparation Metric of Sustainability (SPMS), exhibiting excellent results confirmed the practicality and greenness of the proposed method.

The occurrence of psychotropic drugs in aquatic environments has attracted increasing attention worldwide because of their various ecological toxicities. However, information on their human metabolites remains limited, although some metabolites might retain biological activity and even exhibit stronger biological effects than their parent pharmaceuticals. To accurately characterize the environmental distribution of psychotropic drugs and their human metabolites in aquatic environments, it is necessary to develop analytical methods for their simultaneous determination in environmental waters. In this study, 69 psychotropic drugs and metabolites were selected as target analytes to systematically evaluate their recovery behaviors across different SPE sorbents and develop a reliable extraction method for simultaneous determination of parent compounds and metabolites. The results indicated that the recoveries of 16, 30, and 34 target analytes ranged from 70% to 120% for HLB, MCX, and WAX, respectively. Among them, six hydrophobic and protonatable metabolites exhibited low recoveries on MCX. These low recoveries were associated with metabolic transformation type and modification site, particularly N-demethylation, hydroxylation, and epoxidation, indicating that SPE sorbents may introduce selective recovery bias in metabolite analysis. In addition, HLB and WAX also showed selective recovery differences for specific metabolites, mainly related to insufficient retention of highly polar or structurally modified metabolites. Based on the complementary retention characteristics of different sorbents, an HLB-MCX tandem solid-phase extraction strategy was developed to improve the recovery of structurally diverse psychotropic drugs and their metabolites. It was further applied to determine the psychotropic drugs and metabolites in both wastewater and surface water. Thirteen and eight metabolites were detected in wastewater and surface water, respectively, with concentrations ranging from <LOQ to 214 ng/L and <LOQ to 195 ng/L. These results highlight the importance of incorporating human metabolites into psychotropic drug monitoring and further evaluating their potential environmental risks.

The goal of this study was to investigate cleanup and analytical performance of three different lots of a centrifugal mini-cartridge solid-phase extraction (mini-SPE) product. The study assessed the cleanup of QECh*All extracts from five food matrix types (liquid infant formula, orange, grains, catfish, and kale), followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis of 47 pesticides and contaminants spanning a LogP range of -1 to 8. A second lab used low-pressure (LP)GC- high-resolution (HR)MS with an orbital ion trap (orbitrap) instrument for quantification of the same samples and a separate assessment of sorbents capacity in cleanup. Cleanup of 125 mg equivalent sample in 0.5 mL extract was found to provide >90% more removal of common matrix background components (e.g. fatty acids, cholesterol) than when using more concentrated extracts. Monitoring more than a dozen matrix markers in final extracts within 5 ppm mass accuracy demonstrated that the degree of cleanup generally exceeded the dilution factor, which is an interesting and useful empirical finding for analysts using one-step SPE for cleanup. In GC-MS/MS, an analytical quality control standard (triphenylphosphate) exhibited an RSD of 4.8% (n = 83), while the internal standard (chlorpyrifos-d10) showed a recovery of 97 ± 5% (n = 45). Mean recoveries and RSDs for the targeted analytes were 98% and 8%, respectively, and matrix effects were within ±10% for nearly all analytes. No statistically consistent differences were observed among the three product lots, each containing 20 mg of anhydrous magnesium sulfate (MgSO4) and 12 mg each of primary-secondary amine (PSA) and octadecylsilane (C18) sorbents. Degradation of labile pesticides, such as captan-d6, folpet(-d4), chlorothalonil, and [13]C12-p,p'-DDT, was substantially reduced compared to other methods, with only partial degradation occurring in kale extracts. QECh*All with centrifugal mini-SPE cleanup was also demonstrated to achieve high-throughput in an easy procedure that an individual analyst followed to prepare 75 final extracts in <3 h.

Soybean consumption has been associated with potential health benefits related to the presence of several bioactive compounds, especially isoflavones and soyasaponins. Food processing can drastically modify the phytochemical profile of soybeans, but this complex change cannot be properly investigated by traditional chromatographic techniques. Thus, we aimed at employing two-dimensional liquid chromatography coupled to tandem mass spectrometry to characterize the metabolite profile of soybean meal (SBM) and to study changes caused by fermentation with Saccharomyces cerevisiae (FSBM) and enzymatic hydrolysis with a food-grade cellulase (ESBM). Extraction with methanol:water (1:1, v/v) led to the highest yield and to the most diverse phytochemical profile. Twelve isoflavones were identified in SBM, and both bioprocesses fully converted glycosylated isoflavones to their corresponding aglycones, which were the only isoflavones present in FSBM and ESBM. Among 39 tentatively identified soyasaponins, 28 were unique to SBM, while only three were common to all samples. Fermentation markedly shifted the SBM phytochemical profile: 39 compounds disappeared and 42 were newly detected under these analytical conditions (including 18 soyasaponins). Enzymatic hydrolysis caused a distinct change, with 43 compounds disappearing and 36 new ones emerging (including seven soyasaponins not detected in FSBM). These results highlight the importance of advanced chromatographic methods in unraveling phytochemical complexity and emphasize the role of controlled biotechnological processing strategies in enhancing the chemical diversity of soybean-based ingredients.

Unified chromatographic methods based on modern SFC instrumentation merge supercritical, subcritical and liquid states of mobile phase within a single instrument and a single analytical run, thus providing a high-performance platform for separation of tremendously wide range of analytes. Usually, a gradient of mobile phase starting with almost pure CO2 proceeds to 100% of methanolic-aqueous modifier complemented with various additives. The increased viscosity of the liquid mobile phase limits the applicable flow rates towards the end of the gradient, especially when utilizing modern sub-2 µm particle stationary phases (e.g. UHPSFC columns). In order to stay within the instrument pressure limits and to adjust the mobile phase velocity optimal for chromatographic efficiency, a flow gradient and/or a backpressure regulator gradient can be employed. However, varying flow rate during separation precludes straightforward calculation of retention factors directly from retention times. To address this issue, our paper introduces a procedure for calculating apparent retention factors based on pumped mobile phase volumes continuously corrected for compressibility/expansion. The usefulness of the proposed approach is demonstrated for column screening and comparison of chromatographic selectivity in unified chromatography employing simultaneous gradients of mobile phase composition, mobile phase flow rate, backpressure regulator and makeup liquid flow rate.

A novel urea-based magnetic covalent organic framework composite (Fe3O4@U-COF) was synthesized via a simple, solvent-free mechanochemical method and introduced as a high-performance adsorbent for sample preparation. Comprehensive characterization confirmed a synergistic enhancement in the material's properties. The resulting nanocomposite exhibited a specific surface area (76.05 m[2] g[-1]) significantly greater than its individual components, which is crucial for achieving high adsorption efficiency. The material was successfully applied in a magnetic solid-phase microextraction (MSPME) method for the selective preconcentration of trace Bi(III), Hg(II), Ag(I), and Pb(II) ions. The quantitative determination of the analytes was performed by inductively coupled plasma optical emission spectrometry. Following a systematic optimization of all critical parameters, the optimal conditions were established: an adsorbent amount of 1 mg, a sample volume of 10 mL, and elution with 300 µL of 2 mol l[-1] HCl. Notably, the method demonstrated exceptional robustness, operating effectively across a wide pH range (5-9), which advantageously eliminates the need for sample pH adjustment. Under optimal conditions, the method achieved very low limits of detection (0.03-300 ng l[-1]), wide linear dynamic ranges, satisfactory enhancement factors, and excellent precision, with intra-day relative standard deviations below 2.7%. The practical applicability and accuracy of the method were validated through the analysis of diverse real-world samples, including environmental waters and human plasma, yielding excellent relative recoveries (90-115%) and demonstrating its high tolerance to matrix interferences.

Preparative liquid chromatography (prep-LC) is essential for purifying natural products and pharmaceuticals, but accurate real-time peak detection remains challenging under high-noise, high-flow-rate conditions. Conventional threshold- and derivative-based algorithms often fail to detect shallow, slowly rising peaks because signal changes are masked by baseline fluctuations, and smoothing preprocesses can blur genuine peak features. To overcome these limitations, we developed a moving-window analysis of variance (MW-ANOVA) algorithm that performs an F-test on two consecutive subgroups within a sliding window to distinguish systematic chromatographic trends from random noise. The method requires no peak-shape assumptions or prior denoising. We evaluated MW-ANOVA on analytical-scale symmetric standards, on preparative-scale separation of Houttuynia cordata extract, and on medium-pressure liquid chromatography (MPLC) purification of Lonicera japonica. In the preparative separation, MW-ANOVA reliably detected shallow peaks that were missed by the first derivative method, with a minimum detectable slope of approximately 0.07 mV per data point. It guided automated collection of 28 fractions, three of which showed area percentage purities above 93 % at 254 nm. In the MPLC run, bubble peaks and broad elution profiles were present; MW-ANOVA correctly identified the true elution peaks and ignored bubble peaks, whereas the first-derivative method produced 159 mostly false-positive peaks and the fixed-threshold method failed to define correct boundaries. The algorithm was also robust against gradient-induced baseline drift. MW-ANOVA provides a practical, statistically grounded solution for peak detection in preparative and medium-pressure LC, especially for broad, asymmetric, and low-amplitude peaks.

A novel polymer-based weak cation exchanger functionalized with dicarboxyl groups is described. The stationary phase was synthesized through radical-initiated copolymerization of maleic anhydride and glycidyl methacrylate onto porous poly(styrene-divinylbenzene) microspheres, followed by hydrolysis to yield carboxylate groups. The resultant cation exchanger enables simultaneous isocratic separation of alkali and alkaline earth metals (including NH4[+]) using a single strong acid eluent. High column efficiencies were achieved (e.g., 31,900 and 35,800 plates per meter for Na[+] and K[+], respectively) and high capacity was obtained (4.80 mmol/column or 1.93 μmol/mm[3]). The column also demonstrated good selectivity toward organic amines and other metal ions. The method was successfully applied to the determination of cations in tap water and beer matrices, with recoveries ranging from 92.7% to 109.4%, highlighting its potential for complex sample analysis.