J Sep Sci [JOURNAL]
Sun
200 papers
A Green and Fast Ultra-High Performance Supercritical Fluid Chromatography-Tandem Mass Spectrometry Assay for Sensitive Quantification of Monomethoxy-pentaethylene Glycol Amine in Rat Plasma: Application to Pharmacokinetic Study.
Feng S, Ju H, Guo Y … +7 more , Deng Y, Zhang J, Liu J, Zhou X, Xue H, Shi M, Yin L
J Sep Sci · 2026 Apr · PMID 42013006 · Publisher ↗
Here we present a novel, green analytical platform for the sensitive quantification of the short‑chain excipient monomethoxy polyethylene glycol amine, based on ultra-high-performance supercritical fluid chromatography-t...
Here we present a novel, green analytical platform for the sensitive quantification of the short‑chain excipient monomethoxy polyethylene glycol amine, based on ultra-high-performance supercritical fluid chromatography-tandem mass spectrometry. The core innovation is the effective coupling of supercritical carbon dioxide chromatography with the selective monitoring of protonated molecular ions ([M+H]), which collectively enhance sensitivity and minimize matrix interference. This method enables exceptionally rapid analysis, achieving baseline separation within 3.4 min, and demonstrates high sensitivity with a lower limit of quantification of 5 ng/mL in rat plasma. Comprehensive validation confirmed its robustness, precision, and accuracy across the analytical range. Its environmental sustainability and practical utility were quantitatively endorsed by excellent Analytical GREEnness (0.66) and Blue Applicability Grade Index (72.5) scores. As a demonstrative application, the validated strategy was successfully employed in a rat pharmacokinetic study, establishing a fast, green, and reliable analytical framework for the safety evaluation of PEGylated compounds.
N-Doped Graphene Oxide Quantum Dots as a Novel Stationary Phase for Capillary Electrochromatography.
Maliushevska A, Kartsova L, Mereshchenko A
J Sep Sci · 2026 Apr · PMID 41995232 · Publisher ↗
N-doped graphene quantum dots are carbonaceous smart nanomaterials that play a promising role in analytical chemistry methods, especially as sensors, high-performance liquid chromatography, and gas chromatography station...
N-doped graphene quantum dots are carbonaceous smart nanomaterials that play a promising role in analytical chemistry methods, especially as sensors, high-performance liquid chromatography, and gas chromatography stationary phases. However, their application in electromigration methods was limited to the role of absorbing probes for indirect detection or a pseudostationary phase, while their high surface area and functionality present broader opportunities to increase analytical capabilities of the capillary electrochromatography method. The current work explores their ability to form a stationary phase on the inner walls of a fused-silica capillary for the capillary electrochromatography alone and with the following surfactant modification. It reports stability, the working pH range, efficiency, and selectivity of the novel capillary coating obtained with its further surfactant modification using imidazolium ionic liquids. The stationary phase was applied to separate a mixture of neutral and charged analytes, namely aromatic amino acids and steroid hormones, and the coating applicability to real samples was demonstrated with the food supplement analysis.
Using Radial Flow Stream Splitting Columns to Enable High Through-Put High-Performance Liquid Chromatography for the Analysis of Low Molecular Weight Polystyrenes Using UV/Vis and Charged Aerosol Detection.
Karlsen CE, Cravino JA, Li F … +2 more , Soliven A, Shalliker RA
J Sep Sci · 2026 Apr · PMID 41995223 · Full text
Mass-based detectors, such as, the charged aerosol detector are limited to relatively low flow rates since the mobile phase needs to be removed prior to the detection source. A new column technology, referred to as radia...
Mass-based detectors, such as, the charged aerosol detector are limited to relatively low flow rates since the mobile phase needs to be removed prior to the detection source. A new column technology, referred to as radial flow stream splitting (RFS) whereby flow can be separated in the radial cross flow directly within the outlet of the chromatography column, enables separations to be undertaken at high flow rates, with high efficiency, but low volume load to the detector. In this study we show the improvement in the separation performance of low molecular weight polystyrenes using columns fitted with RFS end fittings and dual detection with a charged aerosol detector and a UV detector in parallel. There is a reduction in operation pressure by 30% and then a subsequent increase in analysis time by up to three to fourfold, where the detection source is ordinarily flow rate limited. Subsequently, flow rates up to 8 mL/min were tested with some separations being completed within 4 min.
Starch-Assisted Molecularly Imprinted ZIF-90 for Enhanced Extraction of Baicalin Followed by Chromatographic Determination.
Chen WZ, Chen HN, Zhang Z
J Sep Sci · 2026 Apr · PMID 41995214 · Publisher ↗
This work addresses the challenge of imparting molecular selectivity to metal-organic frameworks (MOFs) by developing a molecularly imprinted polymer based on ZIF-90 (MI-ZIF). Starch was used as a green and multifunction...
This work addresses the challenge of imparting molecular selectivity to metal-organic frameworks (MOFs) by developing a molecularly imprinted polymer based on ZIF-90 (MI-ZIF). Starch was used as a green and multifunctional auxiliary polymer during a room-temperature preparation of MI-ZIF with baicalin as the template. This strategy leverages the hydroxyl groups of starch to orchestrate the formation of specific recognition cavities within the ZIF-90 framework. The well prepared MI-ZIF exhibited high adsorption capacity and good specificity, with an imprinting factor (IF) of 4.1. The MI-ZIF was used to enrich baicalin from complex matrices such as plant extracts and serum samples. It achieved a high adsorption rate of 93.2% for baicalin from a Scutellaria baicalensis Georgi (SBG) root extract, increasing the baicalin purity from 19.7% to 71.3%. As a solid-phase extraction sorbent for the analysis of serum samples, this method showed high sensitivity with a detection limit of 0.9 ng mL and recoveries in the range of 78.2%-97.2%. The results indicate that the MI-ZIF can significantly improve the enrichment efficiency and detection sensitivity of baicalin, providing an efficient and reliable technical means to solve the problems of enrichment, separation, and trace detection of flavonoid compounds in complex samples.
Development of an LC-Benchtop Nuclear Magnetic Resonance at 125 MHz for Use in Stop-Flow Gradient-Based Separations of Pharmaceutical Compounds.
Menard NRC, Ozvat TM, Frost J … +7 more , Price J, Nishimura M, Ferguson JT, Vial GD, Kuzdzal S, Matsubara T, Schug KA
J Sep Sci · 2026 Apr · PMID 41987508 · Publisher ↗
The development of a high-performance liquid chromatography system with a benchtop H nuclear magnetic resonance (NMR) spectrometer for use in stop-flow mode with a gradient separation is described for the first time. Adv...
The development of a high-performance liquid chromatography system with a benchtop H nuclear magnetic resonance (NMR) spectrometer for use in stop-flow mode with a gradient separation is described for the first time. Advancements in permanent magnet-based benchtop NMR technology have resulted in a field strength of 125 MHz for increased sensitivity with the use of an integrated flow cell. The size and accessibility of benchtop NMRs can allow easier access to NMR for many laboratories. Development was focused on analytical scale instrumentation with concentrations and injection volumes commonly seen in this scale. A combination of pharmaceuticals, specifically acetaminophen, caffeine, warfarin, and ibuprofen, which present a wide profile of different NMR spectra, was examined. Separations and subsequent NMR data collection were performed successfully in multiple common protonated solvents, as opposed to high volumes of costly deuterated solvents, to show the versatility of this new application. Each separated compound presented was successfully analyzed in online stop-flow mode within a single run and was repeated across solvent mixtures. All compounds were detected within a minimum signal-to-noise (SNR) ≥ 3 post-separation after NMR spectra were processed in MestReNova. These were then compared to individual samples tested in typical deuterated solvents for H NMR to confirm the success of this approach.
A Sensitive and Selective UHPLC-MS/MS Method Based on Mercaptopropionic Acid Derivatization for Quantification of Juglone in Biological Matrices.
Zheng X, Deng Y, Cheng S … +6 more , Lu D, Wang Y, Tian Z, Xue H, Yin L, Shi M
J Sep Sci · 2026 Apr · PMID 41981898 · Publisher ↗
In this study, a highly sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of juglone, a poorly ionizable naphthoquinon...
In this study, a highly sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of juglone, a poorly ionizable naphthoquinone, in biological matrices. To enhance ionization efficiency, pre-column derivatization was employed using sulfhydryl nucleophiles (β-mercaptoethanol and 3-mercaptopropionic acid) via Michael addition. Derivatization with mercaptopropionic acid significantly improved mass spectrometric response (50-fold increase) and minimized matrix effects compared to mercaptoethanol. The method, validated per FDA guidelines, exhibited excellent linearity (r ≥ 0.995) from 3 to 150 ng/mL, with accuracy within -10.6% to 4.3% and precision between 1.29% and 5.34%. The lower limit of quantification was 3 ng/mL. Extraction recovery and matrix effects ranged from 99.84% to 102.06% and 92.75% to 98.98%, respectively, with derivatives demonstrating good stability. Cellular uptake studies in MCF-7 cells revealed time-dependent but limited intracellular accumulation of juglone, with the majority retained in the culture medium. This robust derivatization-based UHPLC-MS/MS approach provides a reliable tool for investigating juglone's pharmacokinetics and toxicological mechanisms.
Electromembrane Extraction Assisted by a Metal-Organic Framework and Covalent Organic Framework Composite Incorporated Into Hollow Fiber Membranes for Drug Analysis in Biological Samples.
Sheikhi T, Yamini Y, Razmi H … +2 more , Karimi M, Bagheri M
J Sep Sci · 2026 Apr · PMID 41981885 · Publisher ↗
An assisted electromembrane extraction method using a novel composite of metal-organic framework and covalent organic framework incorporated into the wall pores of a hollow fiber membrane is presented. The metal-organic...
An assisted electromembrane extraction method using a novel composite of metal-organic framework and covalent organic framework incorporated into the wall pores of a hollow fiber membrane is presented. The metal-organic framework and covalent organic framework composite combines the high surface area, tunable porosity, and chemical stability of metal-organic framework with the ordered, covalent network of covalent organic framework, providing a synergistic enhancement of extraction performance. In this technique, the composite material dispersed in an organic solvent is immobilized within the porous fiber walls through capillary forces and ultrasonic assistance, improving both the stability and mass-transfer efficiency of the supported liquid membrane. The morphology, structure, and elemental composition of the sorbent are characterized by scanning electron microscopy, energy-dispersive x-ray analysis, and Fourier-transform infrared spectroscopy. Key experimental parameters affecting extraction efficiency are systematically investigated. The developed method demonstrates a linear response range of 2-1000 µg L with limits of detection between 0.6 and 1.5 µg L for selected model drugs. High precision and recoveries are obtained for human plasma and urine samples. Importantly, the approach is not limited to the studied opioids and can be applied to a broader range of ionizable basic pharmaceuticals. Green analytical assessment tools confirm the method's favorable environmental profile.
Estimating the Human Skin Permeation of Neutral Organic Compounds Using Reversed-Phase Liquid Chromatography and Alternative Organic Solvent Modifiers.
Atapattu SN, Temerdashev A
J Sep Sci · 2026 Apr · PMID 41981880 · Publisher ↗
Determination of human skin permeation is necessary in drug development for topical treatments, the cosmetics sector, and occupational hazard evaluation applications. When compared to the in-vitro assay experiments based...
Determination of human skin permeation is necessary in drug development for topical treatments, the cosmetics sector, and occupational hazard evaluation applications. When compared to the in-vitro assay experiments based on Franz cells for the determination of human skin permeation, the estimation methods based on surrogate chromatographic models are less time-consuming and can be easily automated. In this work, we evaluated surrogate chromatographic alternative organic mobile modifiers, acetone, tetrahydrofuran, and 2-propanol, for the estimation of human skin permeation of small neutral organic compounds using surrogate chromatographic systems. Solvent systems comprising 70% (v/v) acetone and 70% (v/v) 1:1 tetrahydrofuran: 2-propanol on a Kinetex C18 stationary phase demonstrated the best results for the construction of surrogate chromatographic models with prediction errors of 0.180-0.231 and 0.214-0.254 log units, respectively. Proposed surrogate chromatographic models could be used as a rapid screening tool to estimate human skin permeation of small neutral organic compounds.
An Efficient Strategy to Separate the Main Ginsenosides From Steamed Panax quinquefolius by a Combination of Consecutive and Online-Storage Inner-Recycling High-Speed Counter-Current Chromatography.
Guo L, Yu J, Zhong K … +6 more , Wang H, Wang Y, Guo Y, Wang X, Zhao L, Feng Q
J Sep Sci · 2026 Apr · PMID 41981874 · Publisher ↗
This study aimed to isolate and purify compounds from seven-steamed and seven-dried Panax quinquefolius L. by employing a novel separation strategy combining macroporous resin column chromatography, high-speed countercur...
This study aimed to isolate and purify compounds from seven-steamed and seven-dried Panax quinquefolius L. by employing a novel separation strategy combining macroporous resin column chromatography, high-speed countercurrent chromatography (HSCCC), and an on-line storage inner-recycling countercurrent chromatography (OS-IRCCC) mode for the first time, which yielded 12 compounds, including 11 ginsenosides and one methyl naphthalene derivative. Steaming processing of P. quinquefolius triggers significant chemical transformations, characterized by the progressive degradation of major ginsenosides and the concomitant formation and marked accumulation of rare ginsenosides. After preliminary fractionation of the crude extract using a D101 macroporous resin column, three fractions (Fractions III, V, and VI) were selected for further targeted separation. Fractions III and VI were separated using the solvent system of dichloromethane/methanol/HO/isopropyl alcohol with different ratios of 6:5:4:2 and 6.2:4.8:4:2, respectively. For fraction V, traditional HSCCC combined with OS-IRCCC technology was adopted, using methyl tert-butyl ether/n-butanol/acetonitrile/HO (4:2:3:7, v/v). Ultimately, 12 compounds were obtained, including ginsenoside Rd (1, 6.2 mg), ginsenoside Re (2, 7.5 mg), ginsenoside Rg (3, 2.3 mg), 20(R)-ginsenoside Rh (4, 8.3 mg), ginsenoside Rb (5, 3.7 mg), rubilactone (6, 6.4 mg), ginsenoside Re (7, 6.3 mg), 20(S)-ginsenoside Rg (8, 12.1 mg), 20(R)-ginsenoside Rg (9, 5.7 mg), damulin B (10, 2.7 mg), ginsenoside Rb (11, 4.3 mg), and ginsenoside Rg (12, 5.1 mg). This research highlights the efficacy of the novel separation technique for isolating and purifying valuable compounds from seven-steamed and seven-dried P. quinquefolius, providing a robust methodological framework for the targeted purification of complex natural products.
Ultrasensitive Determination of Artificial Sweeteners in Environmental Water Samples Through Capillary Electrophoresis Coupled With Capacitively Coupled Contactless Conductivity Detection.
Chou YM, Hsieh MM, Yeh WT
J Sep Sci · 2026 Apr · PMID 41968922 · Publisher ↗
A dual-enrichment method based on efficient sample cleanup offline ion-pair ultrasonic surfactant-assisted dispersive liquid-liquid microextraction coupled with efficient sample stacking through online field-amplified sa...
A dual-enrichment method based on efficient sample cleanup offline ion-pair ultrasonic surfactant-assisted dispersive liquid-liquid microextraction coupled with efficient sample stacking through online field-amplified sample stacking was devised for the concurrent determination of three artificial sweeteners (acesulfame potassium, saccharin, and cyclamate) using capillary electrophoresis (CE) coupled with capacitively coupled contactless conductivity detection. Various parameters that affected the CE separation performance and the enrichment efficiency of ion-pair ultrasonic surfactant assisted dispersive liquid-liquid microextraction and field-amplified sample stacking were optimized, and excellent CE separation was attained within 20 min. The optimal ion-pair ultrasonic surfactant-assisted dispersive liquid-liquid microextraction method used 240 µL of chlorobenzene as the extraction solvent, 125 µM N-methyl-N, N-dioctyloctan-1- ammonium chloride as the surfactant, and a pH of 7. Sample stacking was achieved by field amplified sample stacking in a 65 mM butyric acid buffer (pH 2.95), and the signal was detected by capacitively coupled contactless conductivity detection (2 Vpp, 200 kHz). Under the optimized conditions, the proposed method achieved enrichment factors ranging from 3158 to 3261. The linear concentration range was found to be 0.3-30 nM (r > 0.9993). The limits of detection for the three artificial sweeteners were in the range of 0.092-0.095 nM. Good precision was verified by the low relative standard deviations for migration times (3.0%) and peak areas (9.0%). The method was successfully applied to spiked environmental water samples. The relative errors for accuracy ranged from -3.4% to 7.5% for lake water, -8.8% to -0.3% for river water, and -6.8% to 9.6% for groundwater. Consequently, this method represents an efficient, rapid, and low-cost technique for extracting and determining artificial sweeteners in environmental water matrices.
Identification of Novel Epimer Metabolites of Sweroside in Rats With Liquid Chromatography/Time of Flight Mass Spectrometry Combined With Picolinoyl Derivatization.
Xu Y, Tang S, Sun Y … +11 more , Li H, Wang Y, Li X, Zhang M, Ma R, Li X, Xu H, Wang X, Wang Y, Zhang H, Wang Z
J Sep Sci · 2026 Apr · PMID 41968906 · Publisher ↗
Sweroside (SRD) is an important iridoid compound that exhibits various pharmacological and biological activities. However, it has been suggested that the remarkable pharmacological activity is due to the metabolites of t...
Sweroside (SRD) is an important iridoid compound that exhibits various pharmacological and biological activities. However, it has been suggested that the remarkable pharmacological activity is due to the metabolites of the iridoids rather than themselves. Therefore, it is crucial to identify the actual bioactive metabolites of iridoids in vivo. The in vivo metabolites of SRD were analyzed using ultra-high performance liquid chromatography-time of flight-mass spectrometry (UHPLC-TOF-MS) and picolinoyl derivatization. Two major exosomatic SRD metabolites derived from human intestinal bacteria, epinaucledal and naucledal, were incubated with liver microsomes, which were reduced to swerosimetabolin A (SMA) and a novel metabolite named swerosimetabolin B (SMB), respectively. Pharmacokinetic studies of SRD were performed Wistar rats. The mass detection sensitivity of the epimer metabolites SMA and SMB using UHPLC-TOF-MS increased apparently following picolinoyl derivatization. These two metabolites distinctly existed within the rat plasma after gavage administration of SRD. The concentrations of SMA and SMB in plasma were analyzed using UHPLC-TOF-MS in positive ion mode, with 3,4-dihydro-5-(hydroxymethyl) isochroman-1-one as an endogenous reference. SMA and SMB achieved maximal mean plasma concentrations of 61.98 ± 8.6 ng/mL (within 2 h) and 42.64 ± 9.6 ng/mL (within 4 h), respectively, following gavage administration of 20 mg/kg SRD. The area under the concentration-time curve for SMB was 1.5-fold higher than that of SMA. Furthermore, the SRD metabolism to the sulfate of SMA and SMB was deduced. This study reports the metabolism of SRD into SMA and SMB in vivo. These metabolites demonstrated high pharmacological activity, which can help clarify the effects of SRD.
Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry Coupled with Multivariate Analysis Deciphers the Dynamic Transformation of Ginsenosides and the Enhancement of Antioxidant Activity in Nine-Steaming-Nine-Drying Black Ginseng.
Lu H, Geng Y, Jing Y … +5 more , Wang S, Gao W, Cai G, Guo Y, Gong J
J Sep Sci · 2026 Apr · PMID 41968896 · Publisher ↗
Black ginseng, produced through the traditional nine-steaming-nine-drying process, exhibits enhanced pharmacological activity compared with unprocessed ginseng. This study investigated the dynamic changes in ginsenoside...
Black ginseng, produced through the traditional nine-steaming-nine-drying process, exhibits enhanced pharmacological activity compared with unprocessed ginseng. This study investigated the dynamic changes in ginsenoside composition and antioxidant activity during steam-drying using a non-targeted metabolomics approach based on ultra-high-performance liquid chromatography-mass spectrometry. A total of 73 ginsenosides were identified, among which 47 differential markers were screened by principal component analysis and partial least squares discriminant analysis. The relative quantification of 29 key markers revealed distinct transformation patterns of ginsenosides during processing. Antioxidant capacities, evaluated by 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), and Ferric Reducing Ability of Plasma (FRAP) assays, showed trends consistent with changes in total ginsenoside content. The results elucidate the transformation pathways of major ginsenosides during steam-drying and provide a theoretical basis for process optimization and quality control of black ginseng-derived products.
Prediction and Analysis of Quality Markers for Hedysari Radix Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry, Network Pharmacology, Molecular Dynamics, and Chemometrics.
Luo X, Li X, Qi P … +1 more , Li C
J Sep Sci · 2026 Apr · PMID 41940669 · Publisher ↗
To establish a scientific and reliable quality evaluation method for Hedysari Radix (HR), this study investigated changes in chemical components before and after the "Rubbing strip" (RS) was processed based on Quality Ma...
To establish a scientific and reliable quality evaluation method for Hedysari Radix (HR), this study investigated changes in chemical components before and after the "Rubbing strip" (RS) was processed based on Quality Marker (Q-Marker) theory. By integrating ultra-high-performance liquid chromatography-tandem mass spectrometry quantitative analysis, network pharmacology, molecular dynamics, and chemometrics to elucidate compositional variations and predict potential quality markers systematically. Results showed significantly increased contents of formononetin, calycosin, liquiritigenin, and γ-aminobutyric acid after the RS was processed, while the contents of ononin, calycosin-7-glucoside, and vanillic acid were reduced. These findings suggest that RS processing may facilitate the transformation of isoflavone glycosides into aglycones through mechanochemical effects. Network pharmacology analysis further identified 10 immune-related compounds, including medicarpin, calycosin-7-glucoside, calycosin, ononin, and formononetin, as potential quality markers. Using chemometric methods such as hierarchical cluster analysis, principal component analysis, and Orthogonal partial least-squares discriminant analysis, robust discrimination models distinguishing RS from "Non-rubbing strip" samples were successfully developed based on the identified Q-Marker. This study, integrating chemical profiling and bioinformatics approaches, identified and predicted potential Q-Markers for HR, providing theoretical and methodological support for establishing a robust and scientifically quality evaluation system for HR.
Integrating Ultra-High-Performance Liquid Chromatography-Photodiode Array Detector Coupled to Quadrupole Time-of-Flight Mass Spectrometry, Network Pharmacology, and Antidepressant Effects to Determine the Optimal Cultivation Years for Paeonia lactiflora Roots.
Jin Z, He HQ, Zhang LP … +2 more , Yu M, Zou ZM
J Sep Sci · 2026 Apr · PMID 41940663 · Publisher ↗
Paeoniae Radix Alba (called Baishao in China, BS), the dried root of Paeonia lactiflora Pall., is a key ingredient in classic Chinese medicine formulas used to treat depression. This study aimed to optimize its cultivati...
Paeoniae Radix Alba (called Baishao in China, BS), the dried root of Paeonia lactiflora Pall., is a key ingredient in classic Chinese medicine formulas used to treat depression. This study aimed to optimize its cultivation period by integrating analysis of chemical constituents and antidepressant effects. Chemical profiling of 5-year-old roots (BS_5Year) using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry led to the identification of 44 major constituents. Network pharmacology analysis predicted that 12 primary constituents, five monoterpenes (oxypaeoniflorin, albiflorin, paeoniflorin, 6'-O-galloylpaeoniflorin, and benzoylpaeoniflorin) and seven polyphenols (gallic acid, methyl gallate, catechin, 1,3,6-trigalloylglucose, 1,2,3,6-tetragalloylglucose, benzoic acid, and 1,2,3,4,6-pentagalloylglucose) were key contributors to the antidepressant effects. Quantitative analysis by ultra-high-performance liquid chromatography-diode array detection showed that the content of these active constituents in BS_5Year was comparable to that in 4-year-old roots (BS_4Year). Behavioral tests (forced swimming and tail suspension) confirmed no significant difference in antidepressant efficacy between the two groups. These findings indicate that a 4-year growth period is sufficient to achieve the desired quality, thus establishing it as the optimal harvest time. Therefore, this study not only guides the cultivation of P. lactiflora but also highlights the importance of integrating chemical and bioactivity data to determine the optimal harvest time for herbal medicines.
Unraveling the Oligomer-specific Uptake Dynamics of PEG600 With 9-18 Subunits in HepG2 Cells by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Coupled With Ammonium Adduct Strategy to Enhance Sensitivity.
Tian J, Zheng X, Tang Z … +7 more , Feng C, Liu M, Zhang N, Xue H, You J, Yin L, Shi M
J Sep Sci · 2026 Apr · PMID 41940645 · Publisher ↗
Polyethylene glycol (PEG) has emerged as a predominant synthetic polymer in contemporary drug delivery systems due to its unique physicochemical properties and versatile functionalization capabilities. Deciphering chain-...
Polyethylene glycol (PEG) has emerged as a predominant synthetic polymer in contemporary drug delivery systems due to its unique physicochemical properties and versatile functionalization capabilities. Deciphering chain-length-dependent biosafety and cellular uptake profiles of PEG polymers is vital for optimizing nanomedicine formulations. We established an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methodology employing ammonium adduction for multiplex quantification of PEG600 oligomers (n = 9-18) in human hepatocellular carcinoma (HepG2) hepatocytes. The method demonstrated linear response (R > 0.99) across 10-1000 ng/mL for PEG600 oligomers (n = 9-18). PEG600 oligomers (n = 9-18) internalization in HepG2 cells was quantified by the validated UHPLC-MS/MS method. PEG600 polymers with 9-18 subunits could be absorbed into HepG2 cells with a small quantity (less than 0.04%). The cytotoxicity study suggested that the half-maximal inhibitory concentration of PEG600 on HepG2 cells is 31.42 mg/mL. PEG600 oligomers demonstrated negligible anti-proliferative effects in HepG2 hepatocytes. This study could provide analytical techniques and critical reference data for cytotoxicity and whole cellular uptake profiles of PEG600 polymers with 9-18 subunits in HepG2 cells. This work establishes an analytical framework for assessing the cytotoxicity and cellular uptake kinetics of PEG600 oligomers (n = 9-18) in HepG2 cells.
Comparative Chromatographic Separation of Free Radicals From Natural Products Using Green Organic Solvents.
Liu C, Liu R, Gao F … +3 more , Zhang Y, Dang J, Fang F
J Sep Sci · 2026 Apr · PMID 41937447 · Publisher ↗
A well-designed liquid chromatographic strategy is essential for the reliable characterization and preparative isolation of natural products (NPs). In this study, an integrated "medium-pressure pretreatment followed by 0...
A well-designed liquid chromatographic strategy is essential for the reliable characterization and preparative isolation of natural products (NPs). In this study, an integrated "medium-pressure pretreatment followed by 0.2% formic acid (FA) HO-EtOH high-pressure purification" strategy was employed to achieve the efficient separation of active compounds in Ribes himalense. Guided by the online high-performance liquid chromatography-DPPH assay, Fr111 and Fr112 were recovered from the R. himalense extract by polyamide- and Spherical C18-medium-pressure liquid chromatography. The chromatographic behavior of these fractions was comparatively examined under hydrophilic and reversed-phase conditions using three commonly employed aqueous-organic mobile phases, namely 0.2% FA HO-ACN, 0.2% FA HO-MeOH, and 0.2% FA HO-EtOH. Under the investigated experimental conditions, the 0.2% FA HO-EtOH system exhibited improved peak resolution and separation selectivity for both fractions relative to the other mobile phases. Ultimately, four free radical inhibitors (>95% purity) with flavonoids as the parent nucleus were obtained from Fr111 and Fr112. Their significant in vitro free radical scavenging activity was validated using the DPPH and ABTS assays. In conclusion, the 0.2% FA HO-EtOH system delivered favorable chromatographic performance while reducing solvent toxicity, demonstrating promise for efficient and greener preparative workflows. The proposed strategy offers a practical and scalable approach for the targeted isolation of structurally similar bioactive compounds from NPs in the foreseeable future.
Oriented Immobilization Coupled With Ligand Fishing Strategy for Rapid Discovery of Direct HMGB1 Inhibitors in Taxus wallichiana var. mairei.
Chen X, Liu J, Qu L … +1 more , Wang F
J Sep Sci · 2026 Apr · PMID 41906428 · Publisher ↗
High Mobility Group Box 1 (HMGB1), a non-histone chromatin protein, is a key inflammatory cytokine. Direct HMGB1 inhibitors have gained attention for their therapeutic potential in age-related diseases by blocking the pr...
High Mobility Group Box 1 (HMGB1), a non-histone chromatin protein, is a key inflammatory cytokine. Direct HMGB1 inhibitors have gained attention for their therapeutic potential in age-related diseases by blocking the pro-inflammatory activity of extracellular HMGB1, but remain limited. Here, an immobilized HMGB1 was synthesized in one step using an oriented immobilization approach and applied to efficiently acquire direct HMGB1 inhibitors from a highly complex matrix. The oriented immobilization method employed a Ni -modified metal-organic framework combined with cell lysate containing engineered His-tagged HMGB1, bypassing the need for pre-purified proteins. The resulting immobilized HMGB1 demonstrated high protein loading capacity (110.2 mg/g) while retaining its cytokine activity. The immobilized HMGB1 was employed to rapidly identify its direct inhibitors from the Taxus wallichiana var. mairei twig. The application of this immobilized HMGB1 enabled efficient capture of 11 HMGB1 ligands from this plant. Among the 11 ligands, seven compounds were structurally characterized by HPLC-Q-TOF-MS/MS. Among these compounds, sciadopitysin was selected due to its higher HMGB1 inhibitory activity and predicted binding affinity to HMGB1. Through fluorescence quenching and surface plasmon resonance analyses, sciadopitysin was experimentally validated, where the surface plasmon resonance data demonstrated direct binding to HMGB1 with a dissociation constant (K) of 2.77 × 10 M, and the observed intrinsic fluorescence quenching provided additional evidence of the ligand-protein interaction. Furthermore, validation in RAW264.7 macrophages demonstrated its dose-dependent suppression of HMGB1-stimulated inflammation (IC = 27.25 ± 3.31 µM). Collectively, a highly efficient ligand fishing strategy for identifying direct HMGB1 inhibitors was established. Moreover, our findings illustrated the correlation between Taxus wallichiana var. mairei and HMGB1 inhibition.
Identification and Structural Elucidation of Galloylated B-Type Procyanidins From Rumex obtusifolius L. Using Enzymatic Digestion, Phloroglucinolysis, and High-Resolution Mass Spectrometry.
Ballert S, Gillmeister M, Kabrodt K … +3 more , Griehl C, Rozhon W, Schellenberg I
J Sep Sci · 2026 Apr · PMID 41906417 · Full text
Procyanidins from plant extracts demonstrate a broad spectrum of bioactive potential. In order to establish structure-activity relationships, it is necessary to fully determine the stereochemistry and substitution patter...
Procyanidins from plant extracts demonstrate a broad spectrum of bioactive potential. In order to establish structure-activity relationships, it is necessary to fully determine the stereochemistry and substitution pattern. Phloroglucinolysis and nuclear magnetic resonance (NMR) are conventionally employed for the analysis of galloylated B-type procyanidins (PBgals). However, these methods require substantial quantities of the compound at a high degree of purity. In this work, five dimeric galloylated procyanidins isolated from Rumex obtusifolius L. roots were analyzed using a two-pronged strategy. Initially, identification and structure elucidation were performed through the implementation of a novel miniaturized enzymatic digestion with polygalacturonase to effectively remove gallic acid residues. Subsequently, the remaining degalloylated dimeric procyanidins were compared to procyanidin B1, B2, B3, B5, and B7 reference substances based on their retention times. Thus, the R/S-configurations at the C2 and C3 positions of both monomer units and the interflavan linkage were determined as either C4→C8 or C4→C6 in a single analysis. In addition, the position of galloylation was ascertained on the upper or lower unit via high-resolution MS with collision-induced dissociation (CID) and electron-activated dissociation (EAD) fragmentation. This innovative fragmentation technology has been demonstrated to preserve the labile bonds, such as the ester bond to gallic acid. Hence, it yields novel diagnostic fragments for structure elucidation. In silico fragmentation analysis revealed that the radical fragment m/z 440.0738 only fits galloylation on the upper unit, thereby validating the results of the CID. Furthermore, phloroglucinolysis was equally miniaturized and utilized as an independent method to corroborate the results of enzymatic digestion and LC-MS/MS. Using these methods, the structural configurations of the five isolated substances were successfully and consistently identified as procyanidin B2-3-O-gallate, procyanidin B1-3-O-gallate, B2-3'-O-gallate, procyanidin B7-3-O-gallate, and procyanidin B5-3'-O-gallate. To our knowledge, this is the first time that these PBgals are reported in R. obtusifolius.
Reduction of Matrix Effects in Liquid Chromatography-Tandem Mass Spectrometry by a Dilution Approach for the Analysis of 245 Pesticide Residues in Selected Vegetables.
Jo YJ, Choi JY, Sardar SW … +1 more , Ishag AESA
J Sep Sci · 2026 Apr · PMID 41906405 · Publisher ↗
Matrix effects in liquid chromatography-tandem mass spectrometry remain a significant challenge in pesticide residue analysis. Ion suppression can introduce uncertainty in quantification and adversely affect both precisi...
Matrix effects in liquid chromatography-tandem mass spectrometry remain a significant challenge in pesticide residue analysis. Ion suppression can introduce uncertainty in quantification and adversely affect both precision and accuracy. To address this, sample dilution is an effective mitigation approach. In this study, matrix effects of 245 compounds (pesticides and metabolites) were evaluated across six representative vegetable matrices: garlic, onion, chives, perilla leaf, cucumber, and tomato, under various dilution factors. Results showed that increasing the dilution factor consistently reduced matrix effects in most matrices. For instance, high-effect matrices like garlic and onion, a 10-fold dilution approximately doubled the number of compounds within the "weak" matrix effect range. In contrast, perilla leaves and chives, which had moderate matrix effects, required two-fold and five-fold dilutions, respectively, to eliminate compounds in the "strong" effect range. On the other hand, cucumbers and tomatoes exhibited minimal matrix effects, as over 95% of their compounds were already in the weak range before dilution, so further dilution had a limited impact. Overall, these results demonstrated that dilution substantially reduced matrix effects, particularly in complex matrices. Specifically, optimal dilution factors of tenfold for garlic, onion, perilla leaf, and chives, and five-fold for cucumber and tomato, enabled accurate quantification using solvent-based calibration curves. In cases where matrix effects could not be reduced, the use of stable isotope-labeled internal standards or other matrix mitigation strategies can serve as an effective alternative.
Cold-Induced Phase Separation Coupled With High-Performance Liquid Chromatography-Fluorescence Detection for Determination of Glyphosate, Aminomethylphosphonic Acid, and Glufosinate in Environmental Water.
Huang C, Wang L, Zhang H … +4 more , Tan W, Wu L, Wang S, Ma J
J Sep Sci · 2026 Mar · PMID 41885006 · Publisher ↗
Glyphosate (GLP) and glufosinate (GLU) are among the most widely used herbicides; however, the strong polarity of these molecules, coupled with the absence of chromophores and fluorophores, poses significant challenges f...
Glyphosate (GLP) and glufosinate (GLU) are among the most widely used herbicides; however, the strong polarity of these molecules, coupled with the absence of chromophores and fluorophores, poses significant challenges for their detection. In this study, a simple and highly sensitive analytical method was developed by coupling cold-induced liquid-liquid extraction (CI-LLE) pretreatment with high-performance liquid chromatography-fluorescence detection, enabling the simultaneous determination of GLU, GLP, and its metabolite aminomethylphosphonic acid (they are denoted as GLP-based compounds) in environmental water. CI-LLE employs an acetonitrile/water mixture, which is frozen at -22°C to induce phase separation, resulting in an upper acetonitrile-rich phase and a lower water-rich phase, thereby achieving effective enrichment and purification of the target compounds. The GLP-based compounds enriched in the aqueous phase were subsequently derivatized with 9-fluoromethyl chloroformate to obtain fluorescent derivatives for detection. The parameters for CI-LLE (treatment time, concentration of chelating agents, sample pH, and acetonitrile content) as well as derivatization conditions (including pH, reagent concentration, and reaction time) were systematically optimized. The developed method exhibited excellent sensitivity (with limits of detection in the range of 0.1-0.4 µg L), satisfactory recoveries (81.0%-105.2%), and repeatability (relative standard deviations <10%). Compared to the chromatogram prior to CI-LLE treatment, the significant enhancement in cleanup efficiency enabled accurate and quantitative analysis. In summary, the proposed CI-LLE method provides an efficient and accurate approach for the determination of GLP-based compounds in aqueous samples, highlighting its strong potential for environmental monitoring applications.
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