Phenolic compounds, secondary metabolites, are essential products of plant metabolism. They are characterized by one or more aromatic rings attached to hydroxyl groups and are categorized into phenolic acids, flavonoids, tannins, stilbenes, and lignans. These compounds play critical roles in plants, contributing to pigmentation, astringency, UV protection, and defense against pests and pathogens. Widely distributed in fruits, vegetables, and other matrices, they are also extracted from waste and byproducts of the food production chain, aligning with sustainable practices. Research on phenolic compounds is extensive, driven by their significant health benefits and diverse biological activity. Extraction is the initial and critical step in their study, with efficiency influenced by factors such as the extraction method, plant matrix properties, solvent choice, temperature, pressure, and time. Recent years have seen a surge in studies on both conventional and innovative extraction methodologies, with a growing emphasis on green technologies. This review provides a comprehensive overview of advancements in green sample preparation (GSP) techniques within the framework of green analytical chemistry (GAC). It highlights strategies to minimize environmental impact, including the use of micro-techniques, assisted extraction methods, and eco-friendly solvents from renewable and non-toxic sources. Experimental design methods for optimizing phenolic compound yields are also discussed. Additionally, the review presents tools for assessing the greenness of sample preparation techniques, focusing on their environmental and operational improvements.

Okadaic acid is a fatty acid polyether biotoxin that poses a significant threat to global human health. In this work, a type of heterogeneous microporous organic network films was synthesized using a surface-initiated polymerization method, which was employed to control the growth of microporous organic network onto the bromine-functionalized surface of a polymer film. The resulting microporous organic network films feature a porous structure, a strong hydrophobic surface, excellent chemical stability, a large specific surface area, and abundant adsorption sites. These films were then applied in film-based solid-phase extraction, and showed outstanding extraction performance for okadaic acid. Under optimized conditions, the film-based solid-phase extraction method was combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to establish a new analytical method with high sensitivity and efficiency. The developed method offers a wide linear range (2.0-1000.0 pg mL) with good linearity (r ≥ 0.9998), a low detection limit (0.5 pg mL), and high precision (RSDs ≤ 5.2%, n = 5). This method was further utilized for the determination of okadaic acid in seafood, successfully detecting trace levels of okadaic acid (5.4 and 13.9 pg mL) with satisfactory recoveries (85.4%-105.8%). The results indicate that the microporous organic network films hold great application potential in sample pretreatment, and provide a novel synthesis strategy for microporous organic network films.

An ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous determination of fipronil and its three metabolites (fipronil sulfone, fipronil sulfide, and fipronil desulfinyl) in zebrafish tissues. Protein precipitation was employed for sample preparation, followed by chromatographic separation on a C column. The method demonstrated excellent linearity (r > 0.99) across a range of 0.1-10 ng/mL, with a lower limit of quantification (LLOQ) of 0.1 ng/mL for all analytes. Validation results confirmed acceptable accuracy (-5.0% to 8.9%), precision (0.6%-13.2%), recoveries (86.3%-113.6%), and minimal matrix effects (86.0%-110.6%). When applied to zebrafish exposed to 50 µg/L fipronil for 12 h, the method revealed preferential accumulation of fipronil and fipronil sulfone in liver tissues, while lower levels were detected in muscle tissues. Fipronil sulfide was rarely detected, and fipronil desulfinyl concentrations were below the LLOQ (0.1 ng/mL) in all tested samples. This reliable and sensitive method is well-suited for environmental toxicology studies, pesticide residue monitoring, and ecological risk assessment.

A novel strategy combining XAD-4 macroporous resin pretreatment and online-storage inner-recycling high-speed counter-current chromatography (OS-IRCCC) was developed for efficient separation of multi-polarity constituents from Polygonatum cyrtonema Hua. The innovation lies in the OS-IRCCC mode, which integrates online storage of target fractions via a six-way valve-equipped recycling loop and inner-recycling separation, overcoming limitations of conventional high-speed counter-current chromatography in resolving structurally similar compounds. After resin fractionation, 40%, 60%, and 95% ethanol eluates were separated using methyl tert-butyl ether/n-butanol/acetonitrile/water (1:4:1:5, v/v) solvent system, chloroform/methanol/water (7:6:4, v/v) solvent system (with OS-IRCCC for Compounds 8 and 9), and petroleum ether/ethyl acetate/methanol/water (5:5:4:3, v/v) solvent system, respectively. Thirteen compounds were successfully isolated, including four high-polarity glycosides (1-4), five medium-polarity phenolic constituents (5-9), and four low-polarity terpenes (10-13). This study demonstrates that the established separation method provides an efficient, rapid, and straightforward approach for isolating active constituents from P. cyrtonema Hua.

Acanthopanax senticosus (AS) is a well-known traditional herbal medicine in China that can invigorate the spleen, tonify the qi and kidney, tranquilize the mind, and possess diverse bioactive functions. However, comprehensive and simultaneous analysis of the main active constituents from AS and in vivo metabolites remains underexplored, which severely impedes its further clinical application. In this study, AB-8 macroporous resin column chromatography was used to enrich the AS extract. Furthermore, ultrahigh performance liquid chromatography with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS) and UPLC-Q-Orbitrap MS techniques were utilized to identify and characterize the chemical constituents and in vivo metabolites of AS extract, respectively. As a result, a total of 60 components were characterized in the AS extract, including 33 phenylpropanoids, 13 lignans, 4 coumarins, and 10 other substances. Additionally, a total of 29 prototype components and 226 metabolites were detected. Specifically, 91 metabolites were discovered in plasma, 19 in brain tissue, 111 in urine, and 76 in feces. The metabolic reactions included Phase I reactions (demethylation, deglucose, oxidation, and hydration) and Phase II reactions (methylation, sulfate esterification, glucuronidation, glucose conjugation, glycine conjugation, and acetylation). In conclusion, our findings provided basic data and a method for further investigations into the relationship between the chemical composition and pharmacological effects of AS. These results are valuable for revealing the material basis, illustrating the mechanism of medical action, and guiding the clinical applications of AS.

A simple, accurate, cost-effective, and sensitive analysis method for the determination of vancomycin in plasma samples has been reported using high-performance liquid chromatography-tandem mass spectrometry. UiO-66-NH metal-organic framework was synthesized through a hydrothermal method, 5 mg of this compound was introduced into 5 mL of model solution or pretreated plasma adjusted to a pH of 8. The mixture was vortexed for 1 min and then the sorbent particles were separated by centrifugation. The upper phase of sorbents was removed and the analytes adsorbed were eluted by a 250 µL mixture of deionized water (pH 2) and methanol at a 1:1 ratio, v/v, under vortexing for 4 min from the sorbent surface. After centrifugation, the eluent was analyzed by the determination system. The synthesized nanoparticles underwent characterization using X-ray diffraction, Fourier transform infrared spectrometry, and scanning electron microscopy. To achieve optimal efficacy, optimization was carried out at every stage of the project, encompassing adsorption, extraction, and desorption processes. Approved validation data consisting of method detection limit (22 and 225 ng/mL in deionized water and plasma, respectively) and limit of quantification (73 and 774 ng/mL in deionized water and plasma, respectively), a wide linear range of the calibration curve (73-250 ng/mL and 774-1250 ng/mL in deionized water and plasma, respectively), and low relative standard deviations for interday and intraday precisions (≤ 4.8%) were obtained by the method. The proposed method can be successfully applied in the analysis for the determination of vancomycin in plasma and deionized water samples.

Triazole antifungals are clinically established agents for the prophylaxis and treatment of invasive fungal diseases in patients with hematologic malignancies. To address the requirements of clinical therapeutic drug monitoring, we developed and validated a novel ultra-high-performance liquid chromatography tandem mass spectrometry method for the simultaneous quantification of three triazole antifungals and their metabolites in human plasma: itraconazole (ICZ), hydroxy-itraconazole (ICZ-OH), voriconazole (VCZ), voriconazole N-oxide (VCZ N-oxide), and posaconazole. The five target analytes were successfully separated using a BEH C18 column (2.1 × 50 mm, 1.7 µm) maintained at 40°C, with gradient elution employing mobile phase A (5.0 mM ammonium acetate in water containing 0.1% formic acid) and mobile phase B (100% acetonitrile [ACN]) at a constant flow rate of 0.4 mL/min. A positive ion pattern was chosen for quantification under multiple reaction monitoring. Following the addition of 10 µL of internal standard, 50 µL of plasma underwent protein precipitation with ACN, and the resulting supernatant was diluted for subsequent analysis. Method validation followed Food and Drug Administration guidelines and Chinese Pharmacopoeia regulations, demonstrating acceptable accuracy, precision, matrix effects, recovery, and stability. The calibration curves exhibited excellent linearity over the range of 0.1-10 µg/mL for all five analytes, with correlation coefficients (r) ≥ 0.9962, while the lower limit of quantification and limit of detection were established at 0.1 and 0.03 µg/mL, respectively. The intra- and inter-day coefficients of variation were below 8.7% at all concentration levels, and the accuracy was 90.0%-113.0%. This methodology was successfully applied for TDM of three triazole antifungals and their metabolites in 150 patients with hematologic malignancies. Therefore, the method demonstrates good analytical performance, establishing its reliability for clinical TDM of triazole antifungals.

This paper aims to achieve efficient selective separation and green purification of menthol using green deep eutectic solvents (DESs). Taking peppermint essential oil as the research object, a green process based on in-situ formation of DESs is proposed to achieve efficient purification of menthol. Taking menthol in peppermint essential oil as a hydrogen bond donor (HBD), eight quaternary ammonium salts and quaternary phosphonium salts were screened as hydrogen bond acceptors (HBA), and tetrabutylammonium chloride (TBAC) was determined as the optimal HBA component. The hydrogen bonding between menthol hydroxyl and TBAC anion was confirmed by FT-IR and H NMR characterization. After optimizing the conditions such as HBA type, n-hexane dosage, molar ratio, and vortex number, under the optimal conditions, the extraction recovery of peppermint essential oil reached 88.62% ± 0.82%, and the menthol purity was 91.16% ± 0.93%. Compared with the original peppermint essential oil, the menthol purity was significantly improved. The recycling experiment showed that TBAC has good reusability, a simple and green purification process, and good application prospects.

Residual solvents, or organic volatile impurities, are a potential toxic risk for pharmaceutical products and have been a manufacturers' concern for many years. Moreover, residual solvents can also affect the quality and stability of not only drug substances but also of drug products. The objective of this work is the development and validation of headspace gas chromatographic method for determination of six residual solvents (methanol, ethyl acetate, isopropyl alcohol, triethylamine, chloroform, and toluene) in losartan potassium raw material. Method development evaluated the critical parameters of sample diluent selection (dimethylsulfoxide and water), optimization of headspace conditions (incubation time and temperature), and chromatographic conditions (column temperature ramp speeds and sample split ratio). Validation was carried out in accordance to Brazilian guidelines. Dimethylsulfoxide was selected as the sample diluent, with an incubation time of 30 min at 100°C. The chromatographic determination was performed on a DB-624 capillary column using programmed temperature, running time of 28 min and a split ratio of 1:5. The method proved to be selective, with suitable sensitivity (limits of quantification below 10% of the specification limits determined by the ICH), precise (relative standard deviations ≤ 10.0%), linear (r ≥ 0.999 for all solvents' standard curves), accurate (average recoveries from 95.98% to 109.40%), and robust under the small modifications in the chromatographic conditions. A simple and reliable method was obtained. The analysis of a losartan potassium raw material batch has detected only isopropyl alcohol and triethylamine as residual solvents, indicating that purification processes applied to this active pharmaceutical ingredient production were capable to remove most solvents from synthesis step.

Copper (Cu) is an essential trace element for maintaining normal cellular functions; however, excessive Cu accumulation has been confirmed to induce hepatotoxicity, while the metabolic mechanisms underlying Cu-induced hepatotoxicity remain unclear. In this study, an innovative integrated separation strategy was established, combining hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF/MS), to systematically resolve metabolomic perturbations in CuCl-exposed rat BRL-3A hepatocytes. Based on their complementary separation mechanisms-HILIC enables efficient retention and separation of polar metabolites via hydrophilic interactions, while RPLC separates nonpolar/weakly polar lipid molecules based on hydrophobic interactions-this analytical strategy significantly expanded the coverage of detectable metabolites and improved the reliability of metabolite identification through cross-validation between the two chromatographic platforms. The results showed that a total of 25 metabolites with significant changes were identified when BRL-3A cells were exposed to 50 µM CuCl (with a cell viability of 85%). These changes were mainly enriched in metabolic pathways such as glutathione metabolism (characterized by a significant decrease in the GSH/GSSG ratio, p < 0.01), arachidonic acid (AA) metabolism (a 42% reduction in AA, p < 0.05), and glycerophospholipid metabolism (a 1.8-fold increase in the levels of lysophospholipids [LysoPCs/LysoPEs], p < 0.05). These findings reveal that oxidative stress, membrane structure damage, and energy metabolism imbalance are the core mechanisms of Cu-induced hepatotoxicity. The integrated liquid chromatography-mass spectrometry (LC-MS) analytical framework established in this study not only provides a novel molecular perspective for elucidating the mechanisms of Cu-induced hepatotoxicity but also demonstrates the application potential of advanced complementary separation technologies in the risk assessment of environmental pollutants.

In this study, poly(N-isopropylacrylamide-4-vinylphenylboronic acid) thermosensitive microcryogels with different magnetic amounts were prepared for immunoglobulin G (IgG) purification. After, the prepared magnetic thermosensitive microcryogels were characterized by scanning electron microscopy, Fourier transform infrared attenuated total reflectance spectroscopy, electron spin resonance spectrometer, and optical microscopy. IgG binding capacities were investigated using aqueous IgG solutions prepared at varying concentrations. The maximum IgG binding capacity of poly(N-isopropylacrylamide-4-vinylphenylboronic acid) thermosensitive microcryogels prepared with different magnetic amounts was calculated as 137 mg/g for microcryogels containing 23.0 mg FeO, 72.0 mg/g for microcryogels containing 11.5 mg FeO, 48.6 mg/g for microcryogels containing 5.62 mg FeO, and 39.2 mg/g for microcryogels without FeO at pH 7.4 phosphate solution and 40°C. It was observed that poly(N-isopropylacrylamide-4-vinylphenylboronic acid) thermosensitive microcryogels can be reused ten times. According to the adsorption results, the adsorption of IgG onto the prepared magnetic poly(N-isopropylacrylamide-4-vinylphenylboronic acid) thermosensitive microcryogels fits the Langmuir isotherm model (Q: 147 mg/g, R: 0.9976). After optimizing the experimental conditions, the purity of the eluted IgG was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The developed microcryogels combine thermoreactivity with magnetic properties for efficient IgG purification, offering tunable binding capacities using boronic acid-based affinity.

Inverse gas chromatography was used to investigate the isomer separation capability and surface properties of Leonurus cardiaca L. leaves and stems. The retention diagrams of the selected solvents were plotted between 303.2 and 328.2 K using the inverse gas chromatography technique. The selectivity coefficients (α) of the structural isomers were determined for L. cardiaca L. biomass under conditions of infinite dilution using the retention diagram. Besides, the surface properties of L. cardiaca L. biomass were investigated using inverse gas chromatography at infinite dilution. The dispersive surface energy of the adsorbent was determined using the Schultz, Dorris-Gray, Donnet-Park, and Hamieh methods. Adsorption enthalpy and Gibbs free energy were calculated using this technique. It was determined that the surface of both L. cardiaca L. leaves ( ) and stems ( ) exhibit acidic characteristics.

Polycyclic aromatic hydrocarbons (PAHs), known for their environmental ubiquity and toxicity even at trace levels, were effectively extracted using a novel tetrafluoroterephthalonitrile-cyclofructan 6 cross-linked polymer (TFN-CF6-CP) coating. This coating was synthesized through in situ cross-linking polymerization of a cross-linking agent with cyclic fructose 6 (CF6) on glass fiber surfaces, subsequently applied in fiber-in-tube solid-phase microextraction (fiber-IT-SPME). The developed method coupled with high-performance liquid chromatography (HPLC) enabled online PAHs analysis. The TFN-CF6-CP demonstrated superior PAHs adsorption capacity, attributable to its distinctive structural characteristics and multiple interaction mechanisms. Under optimized conditions (70 mL extraction volume, 2.5 mL min sampling flow rate, 0% acetonitrile content, and 2.0 min desorption time), the online fiber-IT-SPME-HPLC method achieved remarkable performance metrics: Low detection limits from 0.013 to 0.037 µg L, linear ranges of 0.030-10 µg L, and enrichment factors between 895 and 1946. The TFN-CF6-CP exhibits remarkable durability and outstanding reproducibility (RSD ≤ 10.8%). As a result, it is highly suitable for the detection of contaminants in water samples. These findings indicate that the functionalized glass fibers exhibit exceptional adsorption properties, demonstrating significant potential for PAHs pretreatment in diverse aqueous environmental samples.

The analysis of organic pollutants using gas chromatography coupled to mass spectrometer (GC-MS) with electron impact (EI) ionization and liquid-liquid or solid-phase extraction (LLE or SPE) often suffer from a limited selectivity and arduous sample preparation. Therefore, atmospheric pressure GC-MS/MS coupled with Dual Head-Robotic Tool Change (DH-RTC) Prep and Load (PAL), and solid-phase micro extraction (SPME) arrow was investigated for its suitability. Evaluation of the SPME arrow coated with divinylbenzene-polydimethylsiloxane (DVB-PDMS) using 2-nitrotoluene and nitrobenzene showed acceptable intra-day precision (%relative standard deviation [RSD]) of less than 10% and inter-day precision (n = 35) of 10.54% and 12.45% respectively. Initial trials were conducted to assess the suitability of technique for eight multiclass compounds (from early eluting volatile organic compounds [e.g., 1,2-dichlorobenzene] to challenging late eluting polycyclic aromatic hydrocarbons [PAH] [e.g., benzo[ghi]perylene], specification markers [e.g., octafluoronaphthalene, and hexachlorobenzene], labile and toxic analytes [e.g., endosulfan, and 2,3,7,8-tetrachlorodibenzodioxin], and isobaric closely eluting PAHs [phenanthrene, and anthracene]). The precision (%RSD, n = 6) was ranged from 1.68 to 16.57, with peak asymmetry factor between 1.003 (phenanthrene) and 1.520 (anthracene). Further experiments conducted for priority pollutants (United States Environmental Protection Agency (U.S. EPA) 625.1/2016) indicated that the addition of salt significantly enhanced the responses for 28 analytes. The increase in response was ranged from 1.14-fold for Endosulfan II to 40.53-fold for pentachlorophenol. However, the %RSDs for 51 analytes were better without adding salt and for 12 analytes it improved with the addition of salt. Since the responses for 12 analytes are sufficient even without salt and standard deviations are below U.S.EPA 625.1/2016 limits, further trials were conducted without adding salt. Preliminary trials showed high RSDs up to 52.76% for high-boiling compounds, which were reduced by using internal standards (IS) and lowering the analyte load during injection. Dedicated analysis of high-boiling PAHs and selected compounds with deuterated IS achieved acceptable coefficient of determination (r), %recoveries and %RSDs. The evaluation of method greenness (Green Analytical Procedure Index and Analytical Greenness calculator), and practicality (Blue Applicability Grade Index) indicated that this approach is environmentally sustainable and practical, providing an efficient and validated alternative method for wastewater analysis.

Monitoring trace levels of pesticide residues in water remains a major global analytical challenge. We synthesized a bio-derived hydrophobic deep eutectic solvent (DES), [thymol:tert-butylhydroquinone] (Thy:TBHQ), and coupled it with vortex-assisted dispersive liquid-liquid microextraction (DLLME) prior to gas chromatography (GC) with micro electron capture detection (µECD) for pre-concentration of organophosphorus pesticides (OPPs) (phosphamidon, diazinon, chlorpyrifos). DES formation and stability were confirmed by Fourier transform infrared spectroscopy (O-H red shift) and H-NMR (deshielded phenolic signals). Univariate optimization identified acetonitrile (ACN) as a disperser and ACN/DES = 1:1 (v/v), 100 mL sample, 3 min extraction, and pH 2-6 as optimal. The method showed excellent linearity (coefficients of determination = 0.991-0.997), limits of detection (LODs) of 3.44-15.43 ng L and limits of quantification (LOQs) of 10.32-51.38 ng L, all well below the EU 100 ng L per-pesticide limit, with enrichment factors of 13-27. Precision supported routine application (intra-day relative standard deviation [RSD] 1.67%-3.79%; inter-day 5.25%-9.66%). Spiked real samples across environmental waters: tap (≈90%-100% relative recovery), river (≈60%-82%), and seawater (≈54%-66%), each with RSD < 10% and no background residues detected. The DES retained >85% of its extraction performance over ≥9 adsorption-desorption cycles. Satisfactory extraction efficiency is probably due to the synergistic π-π, hydrogen bonding, van der Waals, and hydrophobic interactions between aromatic OPPs and the DES. Overall, the [Thy:TBHQ]-based vortex-assisted DLLME/GC with µECD platform delivers sensitive, precise, and reusable trace analysis with reduced solvent use, offering a green alternative for monitoring pesticides in diverse water matrices.

Trace detection of aspartame molecules is crucial for the safety management of additives in food. We developed a convenient synthetic method to prepare magnetic dummy molecularly imprinted polymers, innovatively using carbon nanotubes as the stick carrier of the adsorptive material, which can not only improve the physicochemical stability of the material, but also allow the magnetic microsphere core to disperse due to the presence of carbon nanotubes, thereby enhancing the adsorption performance of the material. Molecularly imprinted polymers were fixed as the shell on magnetic carbon nanotubes to improve the adsorption capacity of the adsorptive material for aspartame molecules. Aspartame in food was enriched by the developed magnetic solid-phase extraction method, followed by detection using LC-MS/MS. Meanwhile, the adsorption mechanism of aspartame by magnetic dummy molecularly imprinted polymers was discussed. Due to its strong selectivity and molecular recognition ability for aspartame, an ultra-low limit of detection was achieved after treatment with magnetic dummy molecularly imprinted polymers using the magnetic solid-phase extraction method, with a detection limit of 0.001 ng/g. This adsorption material can be reused five times. The developed method showed high applicability in detecting aspartame in various different food samples on the market, indicating that the method can be used for real samples to achieve trace detection of aspartame in food.

Chiral covalent organic frameworks (CCOFs) show great potential for chromatographic separation, due to their abundant chiral recognition sites and unique structures. A novel CCOF core-shell composite, CCOF-301@SiO, was constructed by post-synthesis modification (PSM) and in-situ growth strategies, and the effect of the amount of acetic acid catalyst on the synthesis of CCOF-301@SiO core-shell microspheres was discussed. The prepared CCOF-301@SiO microspheres composite was used as a multifunctional chiral stationary phase (CSP) for HPLC enantiomeric separation under normal-phase (NP) and reversed-phase (RP) modes. The experimental results showed that the CCOF-301@SiO column exhibited excellent separation performance, with 20 chiral compounds being separated in NP mode and 12 chiral compounds in RP mode, including alcohols, esters, ketones, and amines. Compared with the commercially available Chiralpak AD-H column, the CCOF-301@SiO column exhibited good complementarity in enantiomeric separation performance. In addition, the effects of analyte mass, column temperature, and mobile phase composition on the separation were investigated. Good repeatability and stability of the CCOF-301@SiO column for enantioseparation were observed. This study demonstrates that the CCOFs@SiO constructed by PSM and in-situ growth strategies have great potential as an HPLC stationary phase.

The development of virus-like particle (VLP) production processes is often constrained by the extensive number of analytical methods required for their quantification and characterization, as well as the significant labor demands associated with these techniques. Asymmetrical flow field-flow fractionation (AF4) coupled with in-line detectors, such as ultraviolet (UV) and multi-angle light scattering (MALS), presents a promising label-free and rapid approach to simultaneously assess the quantity and quality of VLP samples. While AF4-MALS has been widely applied for bionanoparticle characterization and quantification in final products and process development, the influence of host cell-derived impurities on the outcome of the analysis remains underexplored. This study investigates the impact of host cell-derived impurities, particularly host cell DNA and chromatin, on AF4-MALS-DLS analysis of both unpurified and purified VLP samples, using HIV-1 gag VLPs produced in CHO cells as a model system. Our results demonstrate that DNA, chromatin, and VLPs can co-elute due to their overlapping size distribution, which, if overlooked, may lead to imprecise determination of VLP concentrations in early process samples and inaccurate yield calculations at later stages. Nevertheless, for total particle quantification, AF4-MALS was shown to be a suitable surrogate for nanoparticle tracking analysis, as the 90° light scattering peak area exhibited a strong linear correlation with total particle concentration. This substitution enables faster sample processing and reduces sample volume requirements. Additionally, our findings highlight the importance of particle concentration and method parameter selection, particularly the detector flow rate, when characterizing samples based on hydrodynamic radius (R). Underestimation of R due to high detector flow rates was proposed as the possible explanation for the higher-than-expected shape factors obtained for VLPs. These results emphasize the need for further optimization of AF4 methods to improve the separation of VLPs from host cell impurities and to ensure reliable characterization of bionanoparticles in complex mixtures.

A comprehensive mass balance approach was developed to quantitatively determine the oxytetracycline-free base in oxytetracycline hydrochloride. The measurement uncertainty of the reference material includes the uncertainty component introduced during the preparation process. The structural analogues of the principal components were quantitatively determined by liquid chromatography (HPLC-DAD), moisture was determined by the Karl Fischer method, nonvolatile impurities were determined by inductively coupled plasma mass spectrometry (ICP-MS), residual solvents were determined by headspace gas chromatography, and the content of chloride ions was determined by ion chromatography. The principal component structural analog impurities were qualitatively analyzed by HPLC-MS, and the structural analogue impurities were quantitatively analyzed by the external standard method. The HPLC-DAD analysis results showed that there were 17 impurities in oxytetracycline hydrochloride. Eleven out of the 17 impurities were successfully identified by HPLC-MS. The results show that the content of structural analog impurities in oxytetracycline hydrochloride is 47.23 mg/g, the content of nonvolatile impurities is 0.18 mg/g, the content of volatile impurities is 0.89 mg/g, the content of chloride ions is 65.16 mg/g, and the water content is 89.90 mg/g. The free base content of oxytetracycline in oxytetracycline hydrochloride was 796.6 mg/g, and the expanded uncertainty was U = 9.4 mg/g (k = 2). The method was verified by quantitative nuclear magnetic resonance (qNMR) spectroscopy. The results showed that the content of free base in oxytetracycline hydrochloride was 788.6 mg/g, and the uncertainty was U = 6.1 mg/g (k = 2). The established mass balance method provides a strong framework for the formulation and certification of oxytetracycline reference standards and offers a reliable alternative to traditional quantitative methods. This study establishes a validated method for the characterization and quantification of oxytetracycline hydrochloride, which has potential applications in the quality control and standardization process of the pharmaceutical industry.

The separation and identification of components in complex mixtures is a requirement for any aspect of modern chemistry. The hyphenated technique of liquid chromatography (LC)-nuclear magnetic resonance spectroscopy (NMR) allows the combination of the separation capabilities of a liquid chromatograph with the structure elucidation capabilities of NMR. As this technique has developed, many approaches have been implemented, and this review aims to discuss these while addressing some of the practical considerations. An examination of the supporting strategies for LC-NMR is discussed, such as the development of solvent suppression methods, the design of more specialized NMR cells, or further hyphenation to other instrumentation such as a mass spectrometer. A sampling of relevant pharmaceutical and natural product applications is discussed that serve to highlight the utility of this technique and its relevancy and current status in the field.

In this study, a miniaturized electromembrane-surrounded solid-phase microextraction (EM-SPME) device was integrated into a microfluidic chip. It was employed for the extraction of phenoxyacetic acid herbicides, including 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D), in environmental and food samples. The separation and determination of the analytes was performed by GC-MS. A nanostructured polypyrrole/zirconium-based metal-organic framework/TiCT titanium carbide (MXene) fiber, synthesized electrochemically, was used as the acceptor adsorbent and also electrode. The simultaneous application of an electric field and nanocomposite-based adsorption enabled efficient preconcentration of the analytes within a compact and low-solvent system. Effective parameters on the extraction of the analytes were investigated and optimized. Under optimized conditions, the developed EM-SPME-GC-MS method exhibited limits of detection of 0.3 µg L for both analytes and linear ranges of 1-1000 µg L for MCPA and 2,4-D (R ≥ 0.9934). Intraday and interday RSDs% were below 7.6% and 10.2% respectively, demonstrating satisfactory precision. The method was validated through real sample analysis in river water, cucumber, and rice extracts, achieving recoveries from 89% to 119%. The chip consumes 10 µL of 1-octanol as SLM and 250 µL of methanolic tetrabutylammonium bromide for derivatization per analysis (total < 0.26 mL organic solvent), which is markedly lower than conventional liquid-liquid extraction/solid phase extraction workflows.