J Sep Sci [JOURNAL]
Sun
200 papers
Wavelet Transform for Frequency-Division Multiplex Mass Spectrometry Combined With High-Performance Liquid Chromatography.
Suzuki H, Iiguni Y, Kitagawa S
J Sep Sci · 2026 Mar · PMID 41885003 · Publisher ↗
Frequency-division multiplexing mass spectrometry (FDM-MS) combined with multiple high-performance liquid chromatographs (HPLCs) is a method for increasing analytical throughput in HPLC-MS by enabling the simultaneous an...
Frequency-division multiplexing mass spectrometry (FDM-MS) combined with multiple high-performance liquid chromatographs (HPLCs) is a method for increasing analytical throughput in HPLC-MS by enabling the simultaneous analysis of multiple samples (Analyst, 2019, 144, 2922-2928). In FDM HPLC-MS, chromatograms from each HPLC, modulated at individual frequencies, are observed in a single mass spectrometer as a mixed chromatogram; therefore, extraction processing is essential to obtain each chromatogram. In this study, we investigated a novel signal extraction approach based on the wavelet transform (WT) instead of the previous Fourier transform-based protocol. Application of WT processing to mixed chromatograms produced two-dimensional scalograms, which enabled the resolution of individual chromatograms according to their modulation frequencies while preserving the analytes' elution times. Chromatograms were extracted by tracing signal intensity profiles along selected lines corresponding to each modulation frequency. Although the extracted chromatograms closely matched the original ones, interference peaks were observed in some cases due to broad spectra. Optimization of the complex Morlet wavelet parameters (B = 3.5, C = 1.5) effectively suppressed such interference. It was also found that the signal intensity of chromatograms extracted from the scalogram depended on the modulation frequency. This dependency suggests that quantitative comparison among simultaneously analyzed HPLC results is difficult. However, this issue was resolved by introducing internal standards into each HPLC-MS system, thereby enabling quantitative comparison. Consequently, this fundamental study demonstrated that combining FDM HPLC-MS with WT-based signal processing is effective for the simultaneous analysis of samples, thereby improving analytical throughput.
β-Cyclodextrin Functionalized Covalent Organic Polymer Monolithic Column for Highly Selective Capillary Electrochromatographic Separation.
Chen X, Shao L, Qian W … +4 more , Yao H, Mao H, Zhang W, Zhang S
J Sep Sci · 2026 Mar · PMID 41877542 · Publisher ↗
The synthesis of covalent organic polymers (COPs) with abundant active sites remains challenging. To address this, we developed β-cyclodextrin (β-CD) functionalized COPs through a modular synthesis strategy, which was co...
The synthesis of covalent organic polymers (COPs) with abundant active sites remains challenging. To address this, we developed β-cyclodextrin (β-CD) functionalized COPs through a modular synthesis strategy, which was comprehensively characterized and subsequently employed as a stationary phase for capillary electrochromatographic separations. Compared with the unmodified column, the β-CD-functionalized COP monolithic column exhibited better separation performance for analytes spanning a wide range of physicochemical properties, including monosubstituted benzenes, alkylbenzenes, phenols, isomers, and chiral compounds, achieving baseline resolution > 1.5 for all tested compounds and a maximum column efficiency of 35 013 plates m for chlorobenzene. Experiments and non-covalent interaction analysis indicated that the synergistic effect between the β-CD cavity and aromatic groups enables the β-CD-functionalized COP stationary phase to exhibit multiple interaction modes (e.g., hydrophobic, π …π stacking, and hydrogen bonding), providing a theoretical foundation for the experimental data. Moreover, the column demonstrated good repeatability and stability, with relative standard deviations for intra-day, inter-day, and column-to-column repeatability all below 2.1%. The column was successfully applied to the determination of monosubstituted benzenes in wastewater samples, showing satisfactory linearity (R ≥ 0.998), which confirms its potential for complex sample analysis.
Recent Developments and Applications of Capillary and Microchip Electrophoresis in Proteomics and Peptidomics (2023-2025).
Štěpánová S, Kašička V
J Sep Sci · 2026 Mar · PMID 41877532 · Full text
This review article presents a broad overview of developments and applications of capillary electrophoresis (CE) and microchip electrophoresis (MCE) in peptidomics and proteomics in the period 2023-2025. It deals with va...
This review article presents a broad overview of developments and applications of capillary electrophoresis (CE) and microchip electrophoresis (MCE) in peptidomics and proteomics in the period 2023-2025. It deals with various methodological aspects, such as sample preparation including preseparation and preconcentration, multidimensional separations by on-line or off-line coupled different CE, MCE, and LC techniques, and their hyphenation with MS detection. Innovations in bottom-up and top-down proteomics are reported. Furthermore, application of CE and MCE techniques in various peptidomic and proteomic studies are demonstrated. They embrace monitoring of posttranslational modifications, basic biological and biochemical research, and analysis of clinical and food samples.
Hydroxypropyl-β-cyclodextrin-assisted Acetonitrile Field-amplified Stacking in Capillary Electrophoresis Enabling Ultra-Trace Quantification of Amlodipine Enantiomers in Human Plasma.
Deng H, Gao W, Liu Y … +5 more , Zhang S, Weng Z, Liu J, Fan G, Li J
J Sep Sci · 2026 Mar · PMID 41863434 · Publisher ↗
Amlodipine besylate, a third-generation dihydropyridine calcium channel modulator, is routinely prescribed as a racemic mixture in the clinical management of arterial hypertension and ischemic chest pain. Among its enant...
Amlodipine besylate, a third-generation dihydropyridine calcium channel modulator, is routinely prescribed as a racemic mixture in the clinical management of arterial hypertension and ischemic chest pain. Among its enantiomeric pair, (S)-amlodipine is the pharmacologically active form and is primarily responsible for the antihypertensive effect. Given that both (R)- and (S)-amlodipine circulate at extremely low concentrations following oral administration, a highly sensitive analytical method is required for enantioselective monitoring. In this study, we developed a simple, sensitive, and cost-effective capillary electrophoresis (CE) method for clinical application, which integrates hydroxypropyl-β-cyclodextrin-mediated chiral recognition with acetonitrile-driven field-amplified sample stacking. Chiral resolution of trace amlodipine (AML) present in human plasma samples was performed in an uncoated bare fused silica capillary (effective length 31.2 cm, internal diameter 75 µm), thermostated at 25°C and operated with an imposed potential of 15 kV. The running buffer consisted of borate (6.25 mM) and phosphate (25 mM), adjusted to pH 2.5, containing 30 mg/mL hydroxypropyl-β-cyclodextrin, and ultraviolet monitoring was conducted at 200 nm. This assay was fully validated for the quantitative measurement of each AML enantiomer in plasma. For (R)- and (S)-amlodipine, linear calibration was achieved across the range 0.2475-19.80 ng/mL and met the commonly accepted bioanalytical criteria. Comparative pharmacokinetic evaluation demonstrated no statistically significant differences between the concentration-time profiles obtained with the proposed CE method and those generated by a reference liquid chromatography-tandem mass spectrometry assay, confirming the accuracy and reliability of the developed procedure for routine therapeutic drug monitoring.
Chemometric-guided Comprehensive Two-Dimensional Gas Chromatography Analysis of Aucklandiae radix and Its Formulated Preparations.
Li Y, Ren C, He M
J Sep Sci · 2026 Mar · PMID 41863429 · Publisher ↗
An integrated strategy combining comprehensive two-dimensional gas chromatography with chemometrics was established to characterize Aucklandiae radix (AR, Mu-xiang) and its complex formulations. By employing contour visu...
An integrated strategy combining comprehensive two-dimensional gas chromatography with chemometrics was established to characterize Aucklandiae radix (AR, Mu-xiang) and its complex formulations. By employing contour visualization, intelligent deconvolution, and in-silico retention index prediction, the method effectively resolved severe co-elution, separating at least five components from single peak vertices. A total of 55 characteristic compounds were identified in the AR essential oil. Comparative analysis revealed that AR and its substitute, Vladimiriae radix (Chuan-Muxiang), shared 21 components, while the former possessed 12 unique constituents compared to 47 in the latter. Chemical divergence was even more pronounced in other substitutes. Furthermore, although costunolide is a major component in the herb powder, it constitutes only 0.4% of the essential oil; thus, dehydrocostus lactone (or dihydrodehydrocostus lactone) and 1,8,11,14-heptadecatetraene were selected as quality markers. The application of these markers to Xiangsha-Yangwei pills from eight manufacturers yielded similarity values of 0.22-0.76. This quantitative profiling revealed significant quality inconsistencies, including the absence of critical components, demonstrating the method's utility for the comprehensive quality assessment of traditional formulas.
Multiple Heart-Cut Ion-Exchange Chromatography-Reversed-Phase Liquid Chromatography Platform for Online Desalting and Fractionation of Monoclonal Antibody Charge Variants.
van der Zon AAM, van der Veen C, Cheung M … +3 more , Baerenfaenger M, Somsen GW, Gargano AFG
J Sep Sci · 2026 Mar · PMID 41858189 · Full text
Minor deviations in biopharmaceutical manufacturing processes, such as those used for monoclonal antibodies (mAbs), can introduce structural modifications that alter protein efficacy and safety. Monitoring these changes...
Minor deviations in biopharmaceutical manufacturing processes, such as those used for monoclonal antibodies (mAbs), can introduce structural modifications that alter protein efficacy and safety. Monitoring these changes is critical to ensure product consistency and efficacy. Ion-exchange chromatography (IEC) is widely employed to assess product quality by separating charge variants. However, the use of non-volatile salts in IEC makes direct identification and characterization of these variants difficult. In this study, we developed a multi-heart-cut IEC-reversed-phase liquid chromatography (RPLC) platform that enables the separation, online concentration, desalting, and fractionation of mAb charge variants. The system integrates a 10-port valve and a six-column selector valve, allowing automated collection of up to five charge variants within a single analytical workflow. Moreover, the platform can be directly coupled to mass spectrometry for the characterization of charge heterogeneity and glycosylation. Systematic optimization of the capture and buffer-exchange conditions, including evaluation of stationary phase types and mobile phase compositions, was performed to maximize overall recovery. This platform can also be used for other chromatographic modes, such as hydrophobic interaction chromatography or size-exclusion chromatography. This integrated multiple heart-cut IEC-RPLC platform provides a high-resolution and efficient platform for detailed characterization of mAb charge variants, facilitating the characterization of critical product quality attributes.
Integrated Ultra-High Performance Liquid Chromatography-High-Resolution Mass Spectrometry, Chromatographic Fingerprinting, Chemometric Evaluation, and a "Spider-Web" Model for Quality Marker Identification in Lysionotus pauciflorus Maxim.
Liu C, Wang Y, Xie J … +10 more , Shahid M, Tang L, Shi D, Lu Y, Pan J, Li G, Wang Y, Huang Y, Li Y, Sun J
J Sep Sci · 2026 Mar · PMID 41834475 · Publisher ↗
Lysionotus pauciflorus Maxim. is a traditional Chinese medicinal herb with antitussive, antiasthmatic, and antioxidant activities. However, systematic studies on its quality control methods remain limited, which restrict...
Lysionotus pauciflorus Maxim. is a traditional Chinese medicinal herb with antitussive, antiasthmatic, and antioxidant activities. However, systematic studies on its quality control methods remain limited, which restricts its further development and application. In this study, an integrated strategy combining separation and analytical techniques with chemometric methods was established to screen quality markers related to the antiasthmatic activity of L. pauciflorus. The chemical constituents of the extracts were systematically characterized using ultra-high performance liquid chromatography coupled with Orbitrap high-resolution mass spectrometry, and plasma pharmacochemistry was employed to investigate potential in vivo bioactive constituents. In addition, high-performance liquid chromatography fingerprint profiles of samples from different batches were established. Principal component analysis and orthogonal partial least squares discriminant analysis were applied to identify key differential components. To evaluate antioxidant activity and link it with chemical constituents, radical scavenging assays combined with high-performance liquid chromatography with diode array detection were conducted to identify potential antioxidant-related components. Furthermore, network pharmacology and molecular docking were used to construct a compound-target interaction network, from which candidate components related to antiasthmatic effects were screened. By integrating all the above results, a multidimensional "spider-web model" was constructed, and the regression area of each component was calculated to evaluate its overall contribution. Ultimately, eight quality markers were identified: chlorogenic acid, neochlorogenic acid, nevadensin 5-gentiobioside, nevadensin 5-O-β-D-glucopyranoside, forsythoside B, paraboside B, acteoside, and lysionotin.
The Influence of Temperature on Stationary Phase Retention in Countercurrent Chromatography: Integrating Experiment With an Extended Du's Plot Model.
Zhao P, Yuan M, Liu Y … +5 more , Ignatova S, Zhang H, Wang Y, Hu P, Zhang M
J Sep Sci · 2026 Mar · PMID 41834463 · Publisher ↗
The selection of a biphasic solvent system is the first and most important step for a successful countercurrent chromatography (CCC) separation. The partitioning coefficient of targets and their related impurities on one...
The selection of a biphasic solvent system is the first and most important step for a successful countercurrent chromatography (CCC) separation. The partitioning coefficient of targets and their related impurities on one side, and the stationary phase retention on the other, are among the parameters used for solvent system selection. At constant temperature, the stationary phase retention of a selected solvent system in any CCC instrument is dominated by the rule of Du's plot, which describes the correlation between stationary phase retention and flow rate. In a biphasic liquid system, physico-chemical properties such as density, viscosity, and interfacial tension between two phases are temperature dependent. In this article, the effect of temperature on the retention of several typical solvent systems was investigated using a high-speed CCC (HSCCC) instrument. The results showed that temperature changes could induce density, viscosity, and interfacial tension variation of the biphasic system with consequent changes in the system's hydrodynamic behavior. As the temperature increased, the density and viscosity of the selected solvent system decreased. This led to a variation in the interfacial tension between two phases depending on the solvent system composition. The stationary phase retention increased when the temperature increased for the tested solvent systems. Based on the obtained results, an improved Du's plot was suggested, accounting for the contribution of operating temperature. The prospect of this article provides hands-on strategies for the development of high-performance separation method for HSCCC.
Ultra-Selective Ultra-High-Performance Liquid Chromatography-Triple-Stage Mass Spectrometry Assay for Quantification of Pyraclostrobin in Zebrafish Liver Tissues Via Multistage Fragmentation to Enhance Sensitivity.
Feng C, Guo Y, Sheng Y … +7 more , Liu M, Liu J, Deng Y, Feng S, Xi Y, Shi M, Yin L
J Sep Sci · 2026 Mar · PMID 41816899 · Publisher ↗
Pyraclostrobin, a widely applied methoxyacrylate fungicide, poses considerable risks to aquatic ecosystems due to its environmental persistence and bioaccumulation potential. Understanding its tissue-specific residue pro...
Pyraclostrobin, a widely applied methoxyacrylate fungicide, poses considerable risks to aquatic ecosystems due to its environmental persistence and bioaccumulation potential. Understanding its tissue-specific residue profile in aquatic organisms such as zebrafish is essential for elucidating ecotoxicological mechanisms and evaluating environmental safety. In this study, a novel, rapid, and sensitive ultra‑high‑performance liquid chromatography-triple‑stage mass spectrometry (UHPLC‑MS) method was developed and fully validated for the quantification of pyraclostrobin in zebrafish liver tissue. The assay utilized an MS transition (m/z 388.0 → 194.0 → 164.0), which significantly enhanced selectivity and sensitivity. The method achieved fast separation (3 min per sample) and exhibited excellent linearity from 1 to 300 ng/mL (R ≥ 0.998). Precision (intra‑ and inter‑day coefficient of variation <10.2%), recovery (94.4%-95.4%), and matrix effects (99.2%-108.9%) all met rigorous bioanalytical criteria. This work represents the first application of UHPLC‑MS for strobilurin analysis in aquatic tissues, providing a reliable high‑throughput platform for residue monitoring and mechanistic toxicology research.
Construction of Molecularly Imprinted Polymer Based on Multiple Affinity Strategies for the Enrichment of Dihydromyricetin From Vine Tea.
Zhang L, Chen R, Huang R … +2 more , Zhang X, Li T
J Sep Sci · 2026 Mar · PMID 41814777 · Publisher ↗
A dihydromyricetin molecularly imprinted polymer was fabricated via a multi-affinity synergistic strategy. Specifically, the deep eutectic solvent (choline chloride/methacrylic acid at molar ratio of 1:2) served as a non...
A dihydromyricetin molecularly imprinted polymer was fabricated via a multi-affinity synergistic strategy. Specifically, the deep eutectic solvent (choline chloride/methacrylic acid at molar ratio of 1:2) served as a non-covalent functional monomer, interacting with dihydromyricetin via hydrogen bonds, 3-acrylamidophenylboronic acid acted as a covalent functional monomer to form boronate affinity covalent bonds with dihydromyricetin, and zinc acrylate formed boronate affinity interactions with dihydromyricetin. The synergistic integration of these three components endowed the polymer with a ternary recognition site simultaneously capable of hydrogen bonding, boronate affinity, and metal chelation. After polymerization, the as-prepared dihydromyricetin molecularly imprinted polymer was characterized by scanning electron microscope, energy dispersive spectrometer, Fourier-transform infrared spectrometer, thermal gravimetric analysis, and x-ray photoelectron spectrum. The effects of imprinting conditions, pH of incubation solution, adsorbent amount, selectivity, stability and reusability of dihydromyricetin molecularly imprinted polymer were also investigated. Under optimal adsorption conditions, the maximum adsorption capacity was calculated to be 359.04 mg/g in 240 min, and the imprinted factor was 1.40, aligned better fitted with Freundlich model and pseudo-second-order kinetic model. The better selectivity of dihydromyricetin molecularly imprinted polymer for dihydromyricetin in the presence of other structurally related compounds indicated its robust anti-interference capability. Furthermore, the dihydromyricetin molecularly imprinted polymer was employed as an adsorbent for the selective extraction of dihydromyricetin from vine tea, with the enrichment efficiency from 68.82% to 70.38%. These results demonstrated that the synergistic imprinting strategies could enhanced the affinity of dihydromyricetin molecularly imprinted polymer toward dihydromyricetin, offering a promising approach for the separation and purification of dihydromyricetin from natural products.
Development of a Liquid-Liquid Extraction Based on Deep Eutectic Solvent for the Analysis of Steryl Glucoside in Biodiesel.
Rida'i MTAA, Chumsantea S, Tulaphol S … +1 more , Aryusuk K
J Sep Sci · 2026 Mar · PMID 41808374 · Publisher ↗
The quantification of trace amounts of steryl glucoside (SG) in biodiesel presents a significant analytical challenge due to its low solubility and tendency to crystallize. This study introduces a dispersive liquid-liqui...
The quantification of trace amounts of steryl glucoside (SG) in biodiesel presents a significant analytical challenge due to its low solubility and tendency to crystallize. This study introduces a dispersive liquid-liquid extraction method utilizing a deep eutectic solvent (DES) for the efficient pretreatment and extraction of SG from a palm oil (PO) biodiesel model. The optimized extraction conditions employed a DES composed of choline chloride and ethylene glycol (1:2 molar ratio), combined with isopropanol as a disperser solvent in a 1:1 weight ratio, and vortex-assisted mixing. High-performance size exclusion chromatography coupled with an evaporative light scattering detector was employed for the analysis of SG, using a mobile phase consisting of toluene, tetrahydrofuran, and acetic acid (100:6:0.25, v/v/v). The method demonstrated a limit of detection of 1.92 µg/mL, a limit of quantification of 5.82 µg/mL, and excellent linearity (R = 0.9928). When applied to commercial PO biodiesel (both crude and refined), the method yielded recovery rates ranging from 86.99% to 96.97%. These results demonstrate that dispersive liquid-liquid extraction with DES is an effective and environmentally friendly approach for SG extraction and analysis in biodiesel.
Macroporous Resin-Based Adsorption Separation of Ginsenosides From American Ginseng Fibrous Roots: Process Optimization and Mechanistic Insights.
Zhai D, Liu A, Bai X … +10 more , Wang H, Zhang Q, Zhou J, Li Y, Yang Y, Meng Z, Shang J, Liu N, Hui M, Li P
J Sep Sci · 2026 Mar · PMID 41795710 · Publisher ↗
American ginseng fibrous roots, often discarded as bioprocessing waste, constitute a valuable source of ginsenosides with high recovery potential. This study established a systematic strategy to enrich ginsenosides from...
American ginseng fibrous roots, often discarded as bioprocessing waste, constitute a valuable source of ginsenosides with high recovery potential. This study established a systematic strategy to enrich ginsenosides from the fibrous roots using macroporous adsorption resins and to investigate the adsorption mechanisms. Among the nine resins tested, the non-polar HPD100 resin, characterized by its styrene-divinylbenzene copolymer matrix and high specific surface area, exhibited superior performance, with an adsorption capacity of 194.1 ± 5.8 mg/g and desorption efficiency above 98.0 ± 1.9%. Dynamic column experiments optimized the operating conditions, achieving 74.9 ± 3.0% purity and 84.9 ± 3.7% recovery at a flow rate of 3 bed volumes per hour, 2.3 ± 0.2 mg/mL sample concentration, and 80% ethanol eluent. The resin maintained stable performance over five adsorption-desorption cycles. Kinetic and thermodynamic analyses revealed that adsorption followed a pseudo-second-order model and Langmuir isotherm, with spontaneous and endothermic characteristics. Structural analyses, including scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopy, revealed hydrogen bonding, van der Waals forces, and π-π stacking as key interactions. Molecular dynamics simulations revealed a thermally enhanced binding effect, demonstrating that elevated temperatures strengthen ginsenoside-resin interactions by increasing binding energy even as hydrogen bonding diminishes. This work elucidates the fundamental adsorption mechanism and establishes a theoretical basis for the high-value valorization of American ginseng by-products through a rationally designed, temperature-controlled strategy.
Simultaneous Quantification of Eight Chiral and Achiral Components in Notopterygii Rhizoma et Radix Extract and Rat Plasma Based on Chiral Stationary Phase-HPLC-MS/MS.
Wu H, Tang L, Xu Z … +5 more , Chen Y, Xia J, Zhou M, Zhu Y, Yao Y
J Sep Sci · 2026 Mar · PMID 41795707 · Publisher ↗
A chiral stationary phase-high performance liquid chromatography-tandem mass spectrometry (CSP-HPLC-MS/MS) approach was developed and validated for the first time to quantify quantification of eight components: the enant...
A chiral stationary phase-high performance liquid chromatography-tandem mass spectrometry (CSP-HPLC-MS/MS) approach was developed and validated for the first time to quantify quantification of eight components: the enantiomers of three chiral components notopterol, oxypeucedanin hydrate, oxypeucedanin (in total six configurations), and achiral components nodakenin, imperatorin, isoimperatorin, bergapten, and ferulic acid, in Notopterygii Rhizoma et Radix. By adjusting the types of chiral stationary phase and the composition ratio of the mobile phase, methanol-acetonitrile (75:25, v/v) was selected as the mobile phase (flow rate 0.5 mL/min), and three chiral components notopterol, oxypeucedanin hydrate, and oxypeucedanin were successfully separated into their enantiomers with Chiralpak IG. The method was applied to analyze both plant extracts and rat plasma samples. A considerable variation in content was observed among the eight components in the plant extracts, with their individual concentrations covering a wide range from 1.04 to 20 400 µg/mL (see Section 3 for details). Similarly, their plasma concentrations also spanned from 0.2 to 262.76 ng/mL. The results demonstrated significant differences in the contents of the components. Notably, the chiral components exhibited marked differences between their enantiomers, suggesting that chiral components should be considered in the quality assessment and control of natural products.
Evaluating the Limitations of One-Dimensional High-Temperature Gas Chromatography - FID for Wax Solution Analysis: A Systematic Study.
Okasaki FB, Pinheiro IF, Bizarre L … +1 more , Guersoni VCB
J Sep Sci · 2026 Mar · PMID 41782258 · Full text
Wax deposition in petroleum systems is intrinsically connected with paraffin wax composition, yet their quantitative characterization by one-dimensional high-temperature gas chromatography with flame ionization detection...
Wax deposition in petroleum systems is intrinsically connected with paraffin wax composition, yet their quantitative characterization by one-dimensional high-temperature gas chromatography with flame ionization detection (HTGC-FID), as prescribed by ASTM D5442, remains challenging. In this work, we systematically quantify the main sources of error affecting HTGC-FID analysis of paraffinic systems and benchmark the chromatographic results against differential scanning calorimetry (DSC). The results demonstrate that solvent-based sample preparation leads to nonhomogeneous solutions at the colloidal scale due to paraffin aggregation, introducing significant sampling errors that intensify with increasing molecular weight, whereas an additional error source arises from incomplete volatilization of heavy paraffins in HTGC. A trade-off between loss of detectability at high dilution and aggregation effects at high concentration was observed, impacting quantitative analysis. The absence of reliable retention time patterns for nonlinear paraffins highlights the intrinsic limitations of one-dimensional HTGC-FID for their structural assignment, emphasizing the need for more advanced chromatographic approaches for comprehensive wax characterization.
Total Breakthrough Strategy in Hydrophilic Interaction Chromatography for IP-RPLC × HILIC Separation of Oligonucleotides.
Aebischer MK, Abdurahman Y, Guillarme D
J Sep Sci · 2026 Mar · PMID 41782256 · Full text
Solvent mismatch between the two dimensions is one of the main limitations in two-dimensional liquid chromatography (2D-LC) and presents a significant challenge for method development. Although 2D-LC provides a powerful...
Solvent mismatch between the two dimensions is one of the main limitations in two-dimensional liquid chromatography (2D-LC) and presents a significant challenge for method development. Although 2D-LC provides a powerful means to increase peak capacity for oligonucleotides analysis compared to conventional one-dimensional LC, solvent incompatibility remains a major obstacle that discourages broader development of such methods. In this work, we investigate a technical solution that can be easily implemented and that eliminates solvent mismatch effects during 2D-LC analysis of oligonucleotides. This approach is based on the total breakthrough behavior of oligonucleotides, which is a phenomenon that allows the injection of large volumes into the second dimension (D) without peak distortion. In this work, we showed that under appropriate conditions, oligonucleotides exhibit total breakthrough behavior in HILIC. This behavior in HILIC is particularly advantageous, as the IP-RPLC × HILIC configuration offers improved mass spectrometry (MS) compatibility compared to IP-RPLC or HILIC × IP-RPLC. Assuming an IP-RPLC × HILIC configuration, we systematically investigated the composition of first-dimension (D) fractions and the D-HILIC parameters influencing total breakthrough to identify the key factors promoting this behavior. Our results offer clear guidance for implementing successful IP-RPLC × HILIC conditions that avoid mismatch effects for oligonucleotides analysis while maintaining a high injection volume in the second dimension. This work demonstrates the potential of the total breakthrough strategy for implementing 2D-LC methods with HILIC as the second dimension.
Deep Eutectic Solvent-Enhanced Counter-Current Chromatography Coupled With Ultrafiltration Liquid Chromatography for Targeted Separation of Cyclooxygenase-2 Inhibitors From Magnolia officinalis.
Zhang X, Liu X, Zhao F … +5 more , He F, Lei Y, Li L, Wang Q, Zou D
J Sep Sci · 2026 Mar · PMID 41761454 · Publisher ↗
Magnolia officinals, owing to its significant anti-inflammatory effect, has been widely utilized in the field of traditional Chinese medicine. This study employed ultrafiltration-LC in conjunction with a deep eutectic so...
Magnolia officinals, owing to its significant anti-inflammatory effect, has been widely utilized in the field of traditional Chinese medicine. This study employed ultrafiltration-LC in conjunction with a deep eutectic solvent-enhanced counter-current chromatography method to achieve the separation and purification of three cyclooxygenase-2 inhibitors from M. officinals plant material. First, cyclooxygenase-2 inhibitors were discovered from M. officinals via ultrafiltration-LC. Response surface methodology was further employed to optimize ultrasonic-assisted extraction parameters. It was found the discovered cyclooxygenase-2 inhibitors could be significantly enriched under the optimized conditions. Using the deep eutectic solvent-enhanced counter-current chromatography, three target cyclooxygenase-2 inhibitors were successfully separated with a solvent system composed of n-hexane/ethyl acetate/DES/water (3:4:5:2, v/v/v/v). Ultimately, three cyclooxygenase-2 inhibitors, including 91 mg of honokiol, 41 mg of 8-obovatol, 95 mg of magnolol were obtained from 500 mg sample. The prioritized magnolol was found to exhibit anti-inflammatory activity via NF-κB pathway phosphorylation cascade activation inhibition and NF-κB/p65 nuclear translocation prevention. It demonstrated the integration of ultrafiltration-LC with deep eutectic solvent-enhanced counter-current chromatography enables the efficient separation of bioactive molecules. It not only advanced the academic understanding of the anti-inflammatory active molecules of M. officinalis, but also created preconditions for the subsequent research, development, and clinical application of M. officinalis.
Characterization of Chemical Constituents in Tianma Xingnao Capsule Using Ultra-High Performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Mass Spectrometry.
Gao X, Li X, Wang Y … +4 more , Wang X, Li Y, Bai L, Sun W
J Sep Sci · 2026 Feb · PMID 41742437 · Publisher ↗
In China, traditional Chinese medicine is often used as a dietary supplement for emergency treatment. In order meet the needs of patients, different regions have different methods. From chemistry consists to reasonable c...
In China, traditional Chinese medicine is often used as a dietary supplement for emergency treatment. In order meet the needs of patients, different regions have different methods. From chemistry consists to reasonable compatibilities, it has showed strong professional operation. Tianma Xingnao Capsule has been used for headache, dizziness, decreased memory, and slow reaction, yet its chemical profile remains unclear. This study innovatively integrates UHPLC-Q-TOF-MS technology with UNIFI informatics platform and multi-database (TCMSP, PubChem, ChemicalBook, ChemSpider, Chemsrc) to establish the first comprehensive chemical profile of this formulation. A total of 144 compounds were provisionally characterized based on retention time, exact mass, and mass spectra data, in conjunction with reference substances and related literature, including 26 phenylethanoid glycosides, 27 oligosaccharide esters, 22 flavonoids (15 xanthones), 12 organic acids, 11 triterpenoid saponins, 10 iridoids, 9 phenols and phenolic glycosides, 7 phenylpropanoids, 6 saccharides, 5 nucleosides, 2 amino acids, and 7 others. The proposed compound-specific fragmentation pathways and establishment of a mass spectral library have made contributions to the standardization of traditional Chinese medicine. The results of this study will provide a reference for elucidating the material basis for Tianma Xingnao Capsule's therapeutic effects and quality control of its active pharmaceutical ingredients.
Modification of a Low-Cost Pipetting Robot for Nanoliter Liquid Handling and Autosampling for Liquid Chromatography-Mass Spectrometry.
Axtell NB, Webber KGI, Truong T … +7 more , Lin HL, Sandberg A, Martin S, Xie X, Huang S, Wang C, Kelly RT
J Sep Sci · 2026 Feb · PMID 41742430 · Full text
Automated liquid handling can achieve higher precision for low-volume dispensing than hand-held pipetting while increasing sample processing throughput and avoiding human error. Current commercial liquid-handling platfor...
Automated liquid handling can achieve higher precision for low-volume dispensing than hand-held pipetting while increasing sample processing throughput and avoiding human error. Current commercial liquid-handling platforms range in cost from tens to hundreds of thousands of dollars, which can be prohibitive for smaller research groups. Home-built systems offer customized functionality but require significant technical knowledge and specialized parts. We reduced the burden of developing homebuilt systems by adapting the hardware and software of a low-cost open-source platform, the Opentrons OT-2. The modifications extend accurate pipetting for the OT-2 to the low nanoliter range, enabling sensitive sample preparation. We then augmented the capabilities of this platform by incorporating control of two-position valves and selector valves for autosampling applications. Our modified system is flexible and can be readily configured to meet the needs of novel LC applications. We demonstrated the system for both sub-microliter sample preparation and autosampling for nano-LC-MS applications. The software developed to control the modified system can be adapted to other custom liquid handling and separations platforms.
Bacteriophage Particle Morphology Defines the Efficiency of Steric Exclusion Chromatography in Centrifuge-Free Concentration Protocol.
Mohri M, Antonova A, Dušek J … +7 more , Pickl A, Angleitner JJ, Bondarenko O, Korniienko N, Kharina A, Burketová L, Kalachova T
J Sep Sci · 2026 Feb · PMID 41742427 · Publisher ↗
Bacteriophages have emerged as biological solution to control bacterial outbreaks in agriculture, food production, and healthcare; however, phage isolation and initial purification remain a costly and time-consuming proc...
Bacteriophages have emerged as biological solution to control bacterial outbreaks in agriculture, food production, and healthcare; however, phage isolation and initial purification remain a costly and time-consuming process. In this study, the classic phage purification method containing two centrifugation steps was compared to the alternative, in which low-speed centrifugation for cell debris removal was replaced by microfiltration in combination with filter aid, and high-speed centrifugation for particle precipitation was substituted with steric exclusion chromatography. The efficiency of both approaches in phage recovery, concentration, and purification was assessed on bacteriophages of different morphology (tailed and non-tailed), accumulated on Pseudomonas syringae, Pseudomonas aeruginosa, and Serratia marcescens. The results revealed that the centrifuge-free approach shortened the purification procedure by more than twice and increased the final titer of non-tailed phages up to 100×, resulting in over 90% yield of suspension of sufficient quality for downstream characterization. In the case of tailed phages, only low-speed centrifugation step was successfully replaced, indicating the crucial impact of particle morphology on efficiency of purification by steric exclusion chromatography. The difference in efficiency between isometric and tailed particles highlights the need for tailored method development for the manufacturing of bacteriophage-based preparations.
Enabling Large-Volume Injections in Hydrophilic Interaction Chromatography of Oligonucleotides Through In-Line Mixing.
Verduin J, Tutiš L, Kritsima A … +2 more , Gargano AFG, Somsen GW
J Sep Sci · 2026 Feb · PMID 41736386 · Full text
Hydrophilic interaction chromatography (HILIC) is an attractive separation mode for the analysis of therapeutic oligonucleotides (ONs). ONs are very polar compounds that are commonly dissolved in highly aqueous media, wh...
Hydrophilic interaction chromatography (HILIC) is an attractive separation mode for the analysis of therapeutic oligonucleotides (ONs). ONs are very polar compounds that are commonly dissolved in highly aqueous media, whereas HILIC eluents often comprise a high percentage of organic solvent. This solvent mismatch can cause breakthrough and peak splitting. In this study, we investigated the effects of the sample solvent composition and injection volume on the HILIC separation of nucleobases and ONs, and to what extent an in-line mixer between injector and column can mitigate breakthrough and peak splitting. Using isocratic HILIC with nucleobases as medium-polar, less-retentive test compounds, we illustrated that an injection solvent of 90% water results in peak broadening, which deteriorates with increasing injection volume, leading to serious peak deformations and asymmetries. Here, in-line mixing did not improve peak shapes, as the mixer volume adds to band broadening and the homogenization of the injection solvent with the HILIC eluent does not lead to on-column focusing conditions. When analyzing an ON mixture (15/16/17-mer) dissolved in a weak solvent (30% water) by gradient HILIC, injection volumes could be increased up to 20 µL (5.7% of the column volume) without losing separation performance. However, when the ON mixture was dissolved in 100% water, injection volumes above 2 µL caused severe peak distortion and extensive analyte breakthrough in gradient HILIC. When injecting the ONs in 100% water with a 100-µL in-line mixer, no analyte breakthrough and peak splitting were observed for injection volumes up to 40 µL, while maintaining baseline separation of the ONs. The usefulness of in-line mixing in HILIC was demonstrated by the analysis of a pharmaceutical antisense ON (ASO). Large-volume injection of this ON in 100% water allowed for the separation and detection of the main compound and adjacent minor impurities, without breakthrough or severe peak distortion.
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