J Sep Sci [JOURNAL]
Sun
200 papers
Chromatographic Purification of Complex Natural Products as a Decision Problem: Retention Prediction, Adaptive Optimization, and Experimental Feedback.
Zhao Y, Han Y, Zhou A … +3 more , Peng X, Yang S, Feng P
J Sep Sci · 2026 Jul · PMID 42394197 · Publisher ↗
Chromatographic purification of complex natural products is not only a problem of improving analytical resolution, but also a sequence of decisions involving uncertain targets, variable matrices, limited samples, competi...
Chromatographic purification of complex natural products is not only a problem of improving analytical resolution, but also a sequence of decisions involving uncertain targets, variable matrices, limited samples, competing objectives, and scale-dependent constraints. Data-assisted chromatography can support this process, but current evidence does not yet establish the autonomous purification of crude natural-product extracts. This review reframes the field as decision support for staged purification workflows, including analytical profiling, target localization, fraction enrichment, semi-preparative transfer, preparative isolation, and purity or recovery confirmation. We evaluate how retention prediction, global retention modeling, Bayesian optimization, active learning, reinforcement learning, and feedback-driven method development can support different decisions, and where their current boundaries lie. Particular attention is given to chromatographic decision quality, defined by improvements in relevant outcomes such as elution-window selection, target enrichment, fraction purity, recovery, activity retention, solvent burden, robustness, and preparative transferability. The review distinguishes currently implementable tools, near-term human-supervised workflows, and aspirational autonomous systems. Worked examples and evidence-readiness tables are used to show how prediction, optimization, feedback, and expert review can be connected without overstating the maturity of the field.
A High-Throughput Analytical Approach Using Polyaniline Doped With Oxalic Acid in Thin Film Solid-Phase Microextraction for the Determination of Personal Care Products in Recreational Waters.
Santos AV, Turazzi FC, Carasek E … +1 more , Barra GMO
J Sep Sci · 2026 Jul · PMID 42388039 · Publisher ↗
In this study, a high-throughput analytical methodology was developed based on the thin film solid-phase microextraction (TF-SPME) technique, employing polyaniline (PAni) as the extractor phase for the determination of p...
In this study, a high-throughput analytical methodology was developed based on the thin film solid-phase microextraction (TF-SPME) technique, employing polyaniline (PAni) as the extractor phase for the determination of personal care products in recreational water samples. The compounds investigated included three parabens, methylparaben (MP), ethylparaben (EP), butylparaben (BP), the UV filter and benzophenone (BZP), with detection performed by high-performance liquid chromatography with a diode-array detector (HPLC-DAD). Experimental conditions were optimized using univariate and multivariate approaches, evaluating the effects of the extractor phase, desorption solvent and time, sample pH, extraction time, and NaCl concentration. Optimal extraction conditions involved the use of PAni doped with oxalic acid, desorption in a mixture of ACN:MeOH (50:50) for 30 min, an extraction time of 150 min, sample pH adjusted to 7, and 20% NaCl. Calibration curves were performed in ultrapure water with determination coefficients greater than 0.9973. The limits of detection (LODs) and quantification (LOQs) were 1.5 and 5 µg L, respectively, for all analytes. Intraday precision ranged from 2% to 19%, while inter-day precision varied between 10% and 22%. Method accuracy was assessed through relative recovery experiments in a water sample from a swimming pool, with recoveries ranging from 74% to 109%. The developed method was applied to six recreational water samples. In three of them, the analyte concentrations were above the LOQ, while in the others, the analytes were not found or their concentrations were below the LOD.
Simultaneous Determination of Dechlorane-Related Compounds in Fish Muscle by Microwave-Assisted Extraction Combined With Enhanced Matrix Removal for Lipids Cleanup and Gas Chromatography-Tandem Mass Spectrometry.
Zhu S, Huang K, Pang W
J Sep Sci · 2026 Jul · PMID 42388014 · Publisher ↗
A robust and efficient analytical method was developed for the simultaneous determination of eight dechlorane-related compounds in fish muscle using microwave-assisted extraction combined with enhanced matrix removal for...
A robust and efficient analytical method was developed for the simultaneous determination of eight dechlorane-related compounds in fish muscle using microwave-assisted extraction combined with enhanced matrix removal for lipids cleanup and gas chromatography-tandem mass spectrometry. The method enables effective extraction of hydrophobic analytes from lipid-rich matrices and efficient removal of co-extracted lipids, resulting in improved analytical performance in terms of recovery and reproducibility. Under optimized conditions, the method exhibited good linearity (R = 0.9976-0.9997), low detection limits (method detection limits: 0.021-0.078 ng/g; limits of quantification: 0.067-0.248 ng/g), and satisfactory recoveries (77.6%-90.1%) with satisfactory precision (relative standard deviations: 2.9%-8.5%). Matrix effects were effectively controlled using matrix-matched calibration combined with isotope-labeled internal standards. The method was successfully applied to real fish samples, demonstrating reliable quantification of trace-level dechloranes. Owing to its simplified workflow and improved throughput, the proposed method provides a practical and reliable approach for the routine analysis of dechloranes in high-lipid biological matrices.
Covalent Zwitterionic Peptide-Based Antifouling Coating of the Fused Silica Capillary Applied for CE Separation of Proteins.
Cao Q, Lv S, Wang Y … +3 more , Wang X, Cong H, Yu B
J Sep Sci · 2026 Jul · PMID 42388012 · Publisher ↗
Zwitterionic compounds exhibit strong hydrophilicity, act as hydrogen bond receptors and donors, and form robust hydration layers through ionic solvation, which effectively prevent unwanted protein adsorption. To reduce...
Zwitterionic compounds exhibit strong hydrophilicity, act as hydrogen bond receptors and donors, and form robust hydration layers through ionic solvation, which effectively prevent unwanted protein adsorption. To reduce nonspecific interactions between analytes and the inner surface of the separation compartment and then improve detection sensitivity and separation efficiency, it is essential to modify fused silica capillaries with functional groups. In this study, we synthesized a zwitterionic antifouling peptide (Glu-Lys)Glu using solid-phase peptide synthesis. Then, we bound the peptide to the inner surface of a fused silica capillary using diazo resin as a coupling agent, resulting in stable and excellent antifouling and separation abilities. To evaluate the properties of the coating, we conducted fluorescent protein adsorption and electrophoresis experiments. The obtained results demonstrated that the coating effectively prevented protein adsorption and made it possible to separate five standard proteins in 20 min. Moreover, three proteins of egg white were separated within 15 min. These findings confirmed the very good anti-protein adsorption performance of the peptide-coated capillary and its practical value for various applications.
Pharmacokinetic Study of Five Lead Components of Psoraleae Fructus in Human Serum by UHPLC-Q-TOF-MS and UHPLC-QqQ-MS/MS after Oral Administration of Qing'e Pills.
Li Z, Su J, Xu X … +7 more , Cai X, Zhong K, Zheng Q, Yang L, Wang Z, Li L, Zhang Z
J Sep Sci · 2026 Jul · PMID 42387995 · Publisher ↗
A rapid and reliable ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) strategy was established to characterize the chemical constituents of Qing'e Pills in vitro a...
A rapid and reliable ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) strategy was established to characterize the chemical constituents of Qing'e Pills in vitro and in vivo. In vitro profiling identified 32 compounds in the Psoraleae Fructus (PF) extract, and 30 compounds related to PF were characterized in the Qing'e Pills extract, mainly belonging to coumarins, dihydroflavones, isoflavones, and chalcones. Following a single oral administration of Qing'e Pills to healthy volunteers, nine prototype compounds were detectable in human serum, confirming systemic exposure to multiple constituents. For quantitative evaluation, a sensitive and selective UHPLC-triple quadrupole-tandem MS (UHPLC-QqQ-MS/MS) method was developed and fully validated for simultaneous determination of five representative analytes (psoralen, isopsoralen [IP], bavachin, neobavaisoflavone, and corylin) in human serum, with osalmid as the internal standard. The validated method was applied to a pharmacokinetic (PK) study in 12 female volunteers receiving Qing'e Pills at the Chinese Pharmacopeia-recommended dose (9 g). Among the monitored analytes, only IP met the quantitative criteria and yielded a complete concentration-time profile, indicating low circulating levels of the remaining components at this clinically relevant dose. Isopsoralen was rapidly absorbed (detectable within 0.5 h), reached a maximum concentration of 85.47 ± 28.96 ng/mL at 1.5 h, and exhibited an area under the curve of 619.52 ± 160.25 ng·h/mL with a terminal half-life of 5.06 ± 0.28 h, suggesting limited systemic exposure with moderate elimination. Collectively, this validated UHPLC-QqQ-MS/MS platform provides a practical approach for chemical profiling and human PK evaluation of coumarins and flavonoids from Qing'e Pills.
Passive Blood-Plasma Separation via Constriction-Expansion Geometry in Untreated Paper Microfluidic Devices.
Murugesan N, Kumar A, Aristotle D … +4 more , Arkat S, Saha S, Shanker G, Das SK
J Sep Sci · 2026 Jul · PMID 42363433 · Publisher ↗
Efficient plasma separation from whole blood is an essential preprocessing step in clinical diagnostics, as plasma is preferred for most colorimetric and biochemical tests over whole blood. Traditional separation methods...
Efficient plasma separation from whole blood is an essential preprocessing step in clinical diagnostics, as plasma is preferred for most colorimetric and biochemical tests over whole blood. Traditional separation methods, such as centrifugation, need laboratory facilities and trained personnel, making them unsuitable for point-of-care (POC) diagnostics. Although paper-based microfluidic devices emerged as a promising solution to the above limitations, most existing paper-based devices rely on filtration membranes, chemical functionalization, or both, increasing device complexity and cost. In the present study, membrane- and reagent-free paper-based microfluidic devices have been designed and developed that achieve blood-plasma separation solely through geometric control of the wicking pathway, eliminating the requirement of any external separation aids. Two devices were designed and fabricated embedding constriction-expansion flow path, with and without a localized hydrophobic barrier, to selectively trap red blood cells while allowing plasma to wick through the porous cellulose network. Experimental results demonstrate consistent plasma separation, with a maximum plasma separation efficiency of ∼64% for the optimized design. Further, to elucidate the underlying mechanism, an analytical formulation for capillary transport considering non-Newtonian blood rheology coupled with two-phase numerical simulations was developed. Finally, the reliability of the proposed devices was further validated through protein (albumin) analysis of the separated plasma, which closely matched the clinical laboratory measurements. The present work establishes a simple, low-cost, equipment- and chemical-free strategy for effective blood-plasma separation achieved solely through structural design, while preserving analyte integrity, offering a viable solution for POC diagnostic applications.
Construction of Nitrogen-rich Graphene Oxide/Chitosan Adsorbent for the Removal of Hexavalent Chromium from Wastewater.
Li J, Yang S, Zhou X … +1 more , Guo X
J Sep Sci · 2026 Jul · PMID 42363427 · Publisher ↗
To overcome the limitations of low hexavalent chromium (Cr(VI)) adsorption capacity and poor acid stability in traditional chitosan (CS)-based adsorbents, a nitrogen-rich graphene oxide (N-GO/CS） adsorbent was prepared....
To overcome the limitations of low hexavalent chromium (Cr(VI)) adsorption capacity and poor acid stability in traditional chitosan (CS)-based adsorbents, a nitrogen-rich graphene oxide (N-GO/CS） adsorbent was prepared. Specifically, GO and polyethyleneimine (PEI) were covalently cross-linked onto CS using γ-aminopropyltriethoxysilane and glutaraldehyde. Herein, GO improves the structural stability of CS in acidic solutions, while PEI enhances the adsorption capacity toward Cr(VI). The successful fabrication of N-GO/CS was confirmed by Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy-energy dispersive spectroscopy. The maximum Cr(VI) adsorption capacities of N-GO/CS, based on the Langmuir model, were 537.6 mg/g at 25°C, 568.2 mg/g at 35°C, and 643.6 mg/g at 45°C, surpassing those of most reported CS-based adsorbents. Adsorption experiments indicated that the process was dominated by chemisorption and followed a homogeneous monolayer mechanism. Thermodynamic investigation confirmed that the adsorption process was endothermic (ΔH = 55.98 kJ/mol) and spontaneous (ΔG < 0). Furthermore, interference tests demonstrated that N-GO/CS selectively adsorbed Cr(VI) in the presence of coexisting metal cations and monovalent ions (e.g., Cl and NO ). Cycling experiments indicated that N-GO/CS maintained 70.6% of its initial adsorption capacity after eight consecutive cycles. The primary adsorption mechanism, including electrostatic interactions, reduction of Cr(VI), and coordination of Cr species, was further elucidated. This work provides valuable technical support for the structural design of adsorbents for the removal of Cr(VI) from wastewater.
Identification of Chemical Constituents and Metabolites of Shipi Powder In Vitro and In Vivo Based on Ultra-High-Performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Tandem Mass Spectrometry.
Ma J, Wang X, Qian Q … +4 more , Tian Y, Wang Y, Wang X, Niu L
J Sep Sci · 2026 Jul · PMID 42359494 · Publisher ↗
Shipi powder (SPP) is a classical traditional Chinese medicine prescription used to warm yang, strengthen the spleen, promote qi movement, and eliminate retained fluid. It is widely used in the clinic for the treatment o...
Shipi powder (SPP) is a classical traditional Chinese medicine prescription used to warm yang, strengthen the spleen, promote qi movement, and eliminate retained fluid. It is widely used in the clinic for the treatment of edema caused by various conditions. However, the comprehensive and simultaneous characterization of the major constituents of SPP in vitro and in vivo has remained unclear, which has limited the identification of its active substances and its further clinical application. Therefore, in this study, a chemical composition database of SPP was established. An ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS) method was developed to characterize the constituents of SPP in vitro and in vivo. A total of 217 compounds were identified in vitro, including 57 alkaloids, 8 phenylpropanoid glycosides, 14 lignans, 41 flavonoids, 41 terpenoids, 15 gingerols, 7 lactones, 16 organic acids, and 18 other compounds. Meanwhile, 151 components were detected in vivo, including 93 prototype components and 58 metabolites, in rat plasma, bile, urine, and feces. In summary, this study systematically characterized the components of SPP in vitro and in vivo for the first time. These results provide a useful basis for identifying the pharmacodynamic substances of SPP and for guiding its clinical application.
Dual Hydrogen Bond-Driven Extraction Using Phenolic Non-Ionic Deep Eutectic Solvent: A Strategy for Ultrafast, High-Efficiency Quinolone Antibiotic Analysis in Aquaculture Water.
Shi Y, Zhao H, Huang L … +8 more , Wu S, Qi P, Di S, Wang Z, Liu Z, Xu S, Wang X, Shuai Q
J Sep Sci · 2026 Jul · PMID 42359490 · Publisher ↗
With the expansion of "blue food" aquaculture farming, monitoring non-point source water pollution caused by hydrophilic fishery drugs quinolones was crucial. To solve the difficulty in separating hydrophilic quinolones...
With the expansion of "blue food" aquaculture farming, monitoring non-point source water pollution caused by hydrophilic fishery drugs quinolones was crucial. To solve the difficulty in separating hydrophilic quinolones from water, a phenolic non-ionic deep eutectic solvent (NIDES) was designed and synthesized. It achieved rapid extraction of quinolones by exploiting the dual hydrogen bonds between the hydroxyl group in phenolic NIDES and the carbonyl groups in quinolones. Subsequently, a green analytical method for detecting quinolones in aquaculture water was established by combining liquid chromatography-tandem mass spectrometry. This approach enables the rapid extraction of 18 quinolones from water within 1 min, achieving satisfactory recovery rates and highly sensitive levels of development (0.0051-0.014 µg L). Enrofloxacin was identified as the most frequently detected quinolone in aquaculture water samples. This work provides a theoretical basis for the design and selection of phenolic NIDES, which demonstrate superior characteristics such as greenness, cost-effectiveness, and instrument-compatibility, and provide important technical support for non-point source pollution monitoring of quinolones.
Unveiling the Volatile Chemical Space of Artemisiae argyi Preparations by GC-Q-TOF MS: A Nontargeted Analytical Strategy for Odor, Pharmacological, and Safety Profiling.
You S, Liang S, Wang Z … +3 more , Yan J, Zhang Q, Lv Q
J Sep Sci · 2026 Jun · PMID 42338144 · Publisher ↗
As a representative traditional Chinese medicine in inhalation therapy, the volatile component profile of Artemisiae argyi preparations remains unclear. This study established an integrated analysis method combining odor...
As a representative traditional Chinese medicine in inhalation therapy, the volatile component profile of Artemisiae argyi preparations remains unclear. This study established an integrated analysis method combining odor, pharmacologically active substances, and potential chemical risk substances. Using headspace gas chromatography-quadrupole-time-of-flight mass spectrometry-based nontargeted analysis under 180°C electric moxibustion conditions, 233 volatile components were identified across 30 batches of A. argyi Folium (AAF), moxa wool, and vinegar-processed charred mugwort (VPCM). Orthogonal partial least squares-discriminant analysis identified 60 significantly differential compounds. Thirteen key odorants were identified (relative odor activity value ≥ 1), with major contributors including vanillin, m-cresol, phenylacetaldehyde, and guaiacol. Results showed that AAF and moxa wool contained significantly higher numbers and peak areas of pharmacologically active substances than VPCM, indicating richer active ingredient release and potentially broader pharmacological potential. However, potential chemical risk substances were also significantly higher in AAF and moxa wool, requiring further health risk assessment. These findings emphasize adherence to the "minimum effective dose" principle to avoid excessive exposure. Statistical correlations among pharmacologically active components across samples were also revealed. This study provides scientific evidence for optimizing moxibustion preparations in inhalation therapy and offers references for analyzing unknown volatile components in other traditional Chinese medicines.
Orthogonal Separation of Propolis by Grafted Reversed-Phase × Normal-Phase Two-Dimensional Thin-Layer Chromatography With a Dedicated Image Processing Tool for Quantitative Performance Assessment.
Lu HY, Li MQ, Chen YY … +5 more , Tan YX, Wang SS, Xie SZ, Fang MJ, Qiu YK
J Sep Sci · 2026 Jun · PMID 42338140 · Publisher ↗
This study presents a grafted reversed-phase × normal-phase two-dimensional thin-layer chromatography (RP × NP 2D-TLC) method for the analysis of propolis, a chemically complex natural resin. A dedicated MATLAB-based app...
This study presents a grafted reversed-phase × normal-phase two-dimensional thin-layer chromatography (RP × NP 2D-TLC) method for the analysis of propolis, a chemically complex natural resin. A dedicated MATLAB-based application was developed to enable a systematic evaluation of 2D‑TLC separation performance in terms of positional repeatability, orthogonality, and resolution. The tool adapts a separation space method to assess orthogonality and implements three resolution algorithms specifically designed to accommodate the irregular spot shapes encountered in 2D planar chromatography. Applied to propolis analysis, the orthogonal system successfully separated 29 components, achieving an effective separation area of 44.8%, and 55.2% of the resolved components exhibited high resolution (>1.0). This platform provides a robust and accessible strategy for the rapid qualitative profiling and separation performance assessment of complex natural extracts.
Efficient Extraction of Nicotine From Tobacco Absolute Oil by Ionic Liquid-Decorated Boron Nitride/Poly(N-isopropylacrylamide) Composite Hydrogel.
Long M, Zhang Y, Chen J … +5 more , Zhao X, Huang Y, Zhao Z, Kang L, Zhou L
J Sep Sci · 2026 Jun · PMID 42333950 · Publisher ↗
Nicotine, a tobacco alkaloid with biological activity and unique pharmacological characteristics, holds significant value across pharmaceutical, agricultural, and industrial applications. Given these attributes, there is...
Nicotine, a tobacco alkaloid with biological activity and unique pharmacological characteristics, holds significant value across pharmaceutical, agricultural, and industrial applications. Given these attributes, there is a growing demand for efficient nicotine extraction from tobacco and its by-products. In this study, a novel ionic liquid (IL)-decorated boron nitride/poly(N-isopropylacrylamide) composite hydrogel (IL-BNNs@P) was synthesized for the effective extraction of nicotine from tobacco absolute oil. Scanning electron microscopy and Fourier-transform infrared analyses confirmed the successful integration of IL-functionalized BNNs within the gel's network structure. This hydrogel revealed an adsorption capacity of 81.49 mg·g with 93.5% extraction efficiency. Furthermore, this hydrogel exhibited exceptional selectivity, with distribution coefficients (K) for nicotine (6.27 L·g) being 1-2 orders of magnitude higher than those for co-existing impurities, yielding selectivity coefficients (α) of 62.6-174.9. Additionally, after five consecutive adsorption-desorption cycles, the extraction efficiency showed a slight decrease from 93.5% to 90.0%, demonstrating excellent reusability. Notably, rapid nicotine desorption was achieved at 40°C within 15 min. This strategy streamlines the nicotine extraction process while providing a promising pathway for advancing green chemistry techniques in the tobacco industry and related fields.
Advancing Sustainable Dispersive Liquid-Liquid Microextraction: Methodological Formats and Eco-Friendly Solvents for Heavy Metal Analysis.
Baig JA, Arain MB, Kazi TG … +1 more , Soylak M
J Sep Sci · 2026 Jun · PMID 42290142 · Publisher ↗
Heavy metals are among the most hazardous chemical pollutants, present in trace amounts in various samples. Prior to the quantitative measurement of heavy metals in a sample, an effective sample preparation technique is...
Heavy metals are among the most hazardous chemical pollutants, present in trace amounts in various samples. Prior to the quantitative measurement of heavy metals in a sample, an effective sample preparation technique is usually adopted. Among the various techniques, dispersive liquid-liquid microextraction (DLLME) is regarded as one of the most effective sample preparation strategies for heavy metal preconcentration due to its simplicity, speed, low cost, high extraction recovery, enrichment factor, and minimal solvent use. Various DLLME modes have been introduced, including ultrasound-assisted DLLME, vortex-assisted DLLME, air-assisted DLLME, spray-assisted DLLME, and effervescence-assisted DLLME, to improve extraction efficiency. In addition, traditional organic solvents are being replaced with alternative solvent systems such as supramolecular solvents (SUPRAs), switchable solvents (SSs), and deep eutectic solvents (DESs) to improve the greenness of these DLLME approaches. Based on the benefits of various DLLME approaches and the use of these environmentally friendly solvents, this review briefly presents the extraction mechanisms and applications of various DLLME modes for heavy metal extraction. In addition, the use of the aforementioned alternative solvents in various DLLME approaches is discussed, along with their benefits. Moreover, the review presents a comparative assessment of different DLLME approaches and environmentally friendly solvents and critically examines limitations, including the use of chlorinated organic solvents in air-assisted DLLME (AA-DLLME), the use of organic solvents for analyte-rich phase dilution, and the use of THF. Furthermore, a table summarizes the figures of merit for all DLLME methods reported (2021-2025) for heavy metal extraction.
Novel Strongly Basic Molecularly Imprinted Solid-Phase Extraction Sorbent for Simultaneous Determination of Catecholamines and Their Metabolites in Urine.
Šilaks A, Podjava A, Bernāte L … +2 more , Grebnevs V, Maciej A
J Sep Sci · 2026 Jun · PMID 42290105 · Full text
Catecholamines are important hormones and neuromediators in the human body. Simultaneous determination of both catecholamines and catecholamine metabolites in bodily fluids can help accurately diagnose dangerous health c...
Catecholamines are important hormones and neuromediators in the human body. Simultaneous determination of both catecholamines and catecholamine metabolites in bodily fluids can help accurately diagnose dangerous health conditions like adrenal tumors. However, the biggest obstacle is the selective separation of these compounds from the biological matrix. In this work, we propose a novel, dual-recognition, non-covalent molecularly-imprinted polymer that utilizes strong anion exchange as the source of sorbent-analyte interaction. (4-Vinylbenzyl)trimethylammonium-homovanillyl alcohol anion salt and (4-vinylbenzyl)trimethylammonium-homoveratric acid anion salt served as template/functional monomer complexes for catecholamines and acidic metabolites, respectively. The sorbent was synthesized using precipitation polymerization and studied with batch adsorption experiments, scanning electron microscopy, Fourier-transform infrared spectroscopy, and Brunauer-Emmett-Teller surface area and pore size analysis. The polymer was loaded into cartridges and tested with acidified urine samples. The analytes are deprotonated and adsorbed via sorbent's tetraalkylammonium moiety in hydroxide form, which also neutralizes excess acid, removing the need for pH readjustment (which is often necessary for urine analysis). The imprinted sorbent can also be reused at least four times without performance deterioration. Norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, vanillylmandelic acid, and homovanillic acid were separated and analyzed in a single run using molecularly imprinted solid-phase extraction combined with liquid chromatography-tandem mass spectrometry (MISPE-LC-MS/MS), with recoveries ranging from 69% (epinephrine) to 97% (homovanillic acid). Method's limits of detection, limits of quantitation, linearity, repeatability, trueness, and intermediate precision were evaluated. Limits of quantitation ranged from 0.7 to 6.5 µg/L (for catecholamines and metanephrines) and from 0.12 to 0.2 mg/L (for acidic metabolites). Compared to commercially available weak-cation exchange and hydrophilic-lipophilic balance cartridges, the imprinted sorbent produced stronger catecholamine signals with minimal volume of urine (25 µL). This study successfully demonstrated molecularly imprinted solid-phase extraction workflow for simultaneous separation and quantitation of urinary catecholamines and their basic and acidic metabolites, proving its compatibility with bioanalysis.
Metabolic Profiling of Panax notoginseng Saponins in Rat Feces Under Colitis by UHPLC-QTOF-MS/MS.
Zhou J, Shao L, Liu J … +5 more , Li SF, Huang JM, Zhang W, Hu K, Huang WH
J Sep Sci · 2026 Jun · PMID 42283093 · Publisher ↗
The biotransformation of Panax notoginseng saponins (PNS) is pertinent to the effect of colitis mediated by gut microbiota, which has variant microbes and diversities in inflammatory bowel diseases (IBDs). However, the m...
The biotransformation of Panax notoginseng saponins (PNS) is pertinent to the effect of colitis mediated by gut microbiota, which has variant microbes and diversities in inflammatory bowel diseases (IBDs). However, the metabolic profiles of PNS mediated by gut microbiota are ambiguous under the pathological condition in vivo. Therefore, it is meaningful to explore the metabolic profiling of PNS mediated by gut microbiota under colitis in vivo. In this study, an ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) method was employed to identify, and relatively quantify PNS and metabolites in rat feces with dextran sulfate sodium salt-induced colitis. The results showed that 89 metabolites were identified in the control group, whereas only 39 ones were detected in the colitis model group, indicating a significant reduction in Panax notoginseng saponin metabolites in the model group, particularly in deglycosylated products. Several main metabolites, such as ginsenoside Rb, ginsenoside compound K, protopanaxatriol, and protopanaxadiol, were generated with relatively low yields in the colitis group. Simultaneously, the deglycosylation reactions as the main biotransformation pathways mediated by gut microbiota of PNS were significantly limited in the colitis group. The employed analytical method helpfully demonstrated that gut microbiota dysbiosis in colitis was associated with the metabolism variation of PNS in vivo.
A Robust and Universal LC-MS/MS Method for Determination of N-Nitrosodimethylamine in Pharmaceuticals Using a C30 Column.
An B, Kim U, Seo S … +7 more , Kim J, Jeong C, Jeong W, Ko E, Kim J, Cho HD, Han SB
J Sep Sci · 2026 Jun · PMID 42283086 · Full text
Since the 2018 valsartan recall, the genotoxic impurity N-nitrosodimethylamine (NDMA) has been frequently detected in various pharmaceuticals. Matrix variability often complicates routine testing, requiring customized an...
Since the 2018 valsartan recall, the genotoxic impurity N-nitrosodimethylamine (NDMA) has been frequently detected in various pharmaceuticals. Matrix variability often complicates routine testing, requiring customized analyses. We developed a universal LC-MS/MS method enabling consistent NDMA detection across diverse pharmaceuticals. Separation utilized a C30 column (150 mm × 4.6 mm I.D., 5 µm), offering superior retention and shape selectivity for polar nitrosamines compared to conventional reversed phases. Unlike C18 or pentafluorophenyl phases, which often exhibit limited retention for NDMA, the C30 phase provides exceptional shape selectivity and enhanced hydrophobic interactions. This structural advantage, supplemented by its resistance to phase dewetting under highly aqueous conditions further ensures robust resolution between NDMA and complex drug matrices. The mobile phase consisted of 0.1% v/v formic acid in water and methanol under gradient elution at 1.0 mL/min. Detection used atmospheric pressure chemical ionization in positive ion mode. The method demonstrated a linear range of 2.0-100.0 ng/mL (r > 0.999), with a limit of detection of 1.0 ng/mL and a limit of quantification of 2.0 ng/mL. Validation per International Council for Harmonisation (ICH) guidelines confirmed accuracy (87.7%-115.5%) and precision (coefficient of variation, CV ≤ 10.7%). Application to 30 diverse pharmaceutical products, including sartans and ranitidine, showed robust resolution (> 2.0) between the analyte and active pharmaceutical ingredients (APIs). Notably, NDMA was quantified in historical batches of ranitidine (164.83 ± 5.68 ng/mL), nizatidine (10.25 ± 0.52 ng/mL), and amitriptyline (2.28 ± 0.19 ng/mL). While the histamine type 2 receptor antagonists significantly exceeded the acceptable daily intake limit, the remaining 27 products showed no detectable NDMA. These findings highlight the method's effectiveness for real-world surveillance and the critical risk of post-manufacturing NDMA generation during prolonged storage. This universal C30-based method provides a practical, reliable tool for routine screening, facilitating regulatory compliance and improved patient safety without drugspecific method development.
Impurity Profiling of an Antibody-Antibiotic Conjugate (AAC) Drug-Linker Using Multiple 2D-LC Modes.
Wang J, Zhang K, Goyon A
J Sep Sci · 2026 Jun · PMID 42272334 · Full text
Antibody-antibiotic conjugates (AACs) represent a class of highly potent biopharmaceuticals in which monoclonal antibodies (mAbs) are conjugated to antibiotic payloads via chemical linkers. Characterizing the peak purity...
Antibody-antibiotic conjugates (AACs) represent a class of highly potent biopharmaceuticals in which monoclonal antibodies (mAbs) are conjugated to antibiotic payloads via chemical linkers. Characterizing the peak purity and impurity profile of AAC drug-linkers is analytically challenging due to their complex, heterogeneous structures. In this study, a multimodal two-dimensional liquid chromatography (2D-LC) workflow was developed to assess peak purity and profile impurities in an AAC drug-linker. The strategy integrated comprehensive (LC × LC), heart-cutting (LC-LC), and selective comprehensive (sLC × LC) modes to provide comprehensive impurity characterization. Initially, LC × LC was utilized for rapid reference method screening, successfully identifying co-elution regions without extensive 1D-LC optimization. Targeted LC-LC was subsequently employed to resolve impurities co-eluting with the main peak through independent D optimization. Finally, sLC × LC improved the resolution of two impurities co-eluting within the fronting and tailing regions of the main peak, facilitating their quantification and identification. Beyond increasing peak capacity, this approach provided a rapid route to a comprehensive impurity profile by bypassing exhaustive method development. It directly improved the reliability of peak integration and facilitated the identification of hidden impurities, ensuring a more rigorous determination of the drug-linker purity. The method coupled a polar-selective SB-Aq column with a Phenyl-Hexyl column, effectively resolving impurities associated with both the hydrophilic linker and the hydrophobic drug moiety through 2D-LC. The developed 2D-LC platform provides a highly effective solution for impurity profiling in AACs and is readily adaptable to other bioconjugates, such as antibody-drug conjugates (ADCs) and antibody-oligonucleotide conjugates (AOCs), requiring high-resolution characterization of complex drug-linker species.
Affinity Capillary Electrophoresis for the Study of Biomolecular Interactions: Recent Advances and Future Perspectives.
Jin Y, Zhang D, Wang H
J Sep Sci · 2026 Jun · PMID 42272330 · Publisher ↗
Biomolecular interactions, such as protein-protein, protein-DNA, and protein-RNA interactions, are foundational to numerous vital biological processes and underpin the theoretical basis for disease diagnostics, therapeut...
Biomolecular interactions, such as protein-protein, protein-DNA, and protein-RNA interactions, are foundational to numerous vital biological processes and underpin the theoretical basis for disease diagnostics, therapeutic strategies, and drug discovery. The inherent complexity of these systems-characterized by intricate conformational dynamics and sophisticated regulatory networks-poses significant challenges for analytical instrumentation. Affinity capillary electrophoresis (ACE) has emerged as a powerful technique specifically designed for this purpose, providing a rapid, efficient, and precise means of analyzing proteins and nucleic acids. This review summarizes recent advances in the application of ACE for the study of biomolecular interactions, examines its integration with other separation and analytical techniques, and discusses developmental trends in this dynamic field.
Thermodynamic and Kinetic Parameters of Four Drugs Toward Beta-2 Adrenergic Receptor Were Measured Robustly and Accurately Through a Chromatographic Column With CotB1 Peptide-Mediated Immobilization.
Wang P, Cui N, Xu R … +3 more , Wang J, Ma L, Li P
J Sep Sci · 2026 Jun · PMID 42265944 · Publisher ↗
Thermodynamics and kinetics of drug-protein interactions are key indices for evaluating pharmacological activity during the early stages of drug discovery. In this work, the recombinant beta-2 adrenergic receptor (βAR) w...
Thermodynamics and kinetics of drug-protein interactions are key indices for evaluating pharmacological activity during the early stages of drug discovery. In this work, the recombinant beta-2 adrenergic receptor (βAR) with the fusion of Halo tag or CotB1 peptide (CotB1p) tag was immobilized onto silica gels for preparing the two affinity chromatographic columns. Two experimental models, such as the injection amount-dependent method and nonlinear chromatography, were employed to measure equilibrium constants and rate constants of four drugs, including bambuterol, salbutamol, clorprenaline, and tulobuterol, toward βAR. Compared with nonlinear chromatography, the binding constants measured by the injection amount-dependent method were more comparable to those from the radio binding assay. CotB1p-mediated immobilization yielded larger association constants (K) and the smaller dissociation rate constants (k) of drug-βAR interaction, suggesting that the small-sized CotB1p tag has less interference of protein conformations and the binding sites' microenvironment than the large-sized Halo tag. Thermodynamic analysis revealed that four drugs were spontaneously bound to βAR, and the bindings between them were mainly driven by electrostatic interactions. Our results indicated that the CotB1p-mediated immobilization strategy, resulting in the robust and accurate measurement of the binding parameters of ligand-receptor interaction, will bring new perspectives in applying affinity chromatography in the research fields of drug screening and drug-protein interaction.
Host-Dependent Metabolomic Variations in Viscum coloratum Revealed by UHPLC-Q-TOF-MS/MS and Integrated Annotation Strategies.
Wang J, Chen J, Ju X … +8 more , Liu F, Yu W, Huang L, Qiu J, Jia S, Wang C, Xiang C, Sun Q
J Sep Sci · 2026 Jun · PMID 42261120 · Publisher ↗
Viscum coloratum (Kom.) Nakai, a widely distributed hemiparasitic medicinal plant in Asia, exhibits host-influenced metabolic traits that remain inadequately characterized. To further investigate this issue, an untargete...
Viscum coloratum (Kom.) Nakai, a widely distributed hemiparasitic medicinal plant in Asia, exhibits host-influenced metabolic traits that remain inadequately characterized. To further investigate this issue, an untargeted metabolomics approach was employed to comprehensively delineate host-dependent metabolic variation. Ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS), a powerful analytical platform for untargeted metabolomics, was used to examine samples collected from two host species in Hubei Province, China. Differential metabolites were screened through multivariate chemometric analysis and characterized using an integrated annotation strategy to ensure systematic and reliable identification. Sixty-eight differential constituents were annotated and mainly assigned to phenylpropanoids, polyketides, lipids and lipid-like compounds, and organic acids and derivatives. These classes represented the major metabolic groups associated with host-dependent variation among the samples analyzed in this study. Furthermore, an exploratory 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay revealed significant differences in antioxidant capacity between the two groups, suggesting that host plants may influence the pharmacologically relevant properties of V. coloratum. These findings highlight the importance of considering host factors in its quality control, provide a comprehensive framework for differential metabolite identification, and enhance the understanding of the metabolic characteristics of V. coloratum.
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