J Sep Sci [JOURNAL]
Sun
200 papers
Electrochemistry Coupled to High-Resolution Mass Spectrometry for Phase I and Phase II Metabolic Studies of Bioactive Constituents in Phellodendri Chinensis Cortex.
Yang M, Liu T, Ma W … +4 more , Esmayil A, Yu Z, Du K, Chang Y
J Sep Sci · 2025 Oct · PMID 41073364 · Publisher ↗
An innovative approach combining electrochemistry with online quadrupole time-of-flight-mass spectrometry (EC-MS/MS) was employed to study the oxidative products and metabolic pathways of three bioactive compounds found...
An innovative approach combining electrochemistry with online quadrupole time-of-flight-mass spectrometry (EC-MS/MS) was employed to study the oxidative products and metabolic pathways of three bioactive compounds found in Phellodendri Chinensis Cortex, including phellodendrine, obaculactone, and obacunone. This advanced analytical technique provided detailed insights into their transformation processes. The simulation of phase I metabolism reactions was conducted within an electrochemical microreactor system. The heart of this apparatus was a reliably performing boron-doped diamond electrode. Meanwhile, a defined concentration of glutathione (GSH) was injected into the reaction system to obtain phase II metabolites. The metabolites were collected and concentrated for offline electrochemistry-ultra-high-performance liquid chromatography-quadrupole-time of flight-mass spectrometry analysis (EC-UHPLC-MS/MS). The main converted products and metabolic pathways were further verified through in vitro liver microsome incubation experiments. It was revealed that the simulated metabolic process based on the electrochemical system effectively produced a variety of metabolites from the compounds, which were subsequently compared with those obtained from rat liver microsomal incubations. Without matrix interference, the drug metabolic process could be effectively simulated and analyzed using the electrochemical system. The findings underscore the utility of electrochemistry as a robust tool for preliminary investigations into the metabolism of natural products, offering a reliable and efficient alternative to traditional methods.
Quantifying Biomolecular Interactions in High-Conductivity Samples With Capillary Electrophoresis.
De Silva M, Aruna S, Samarakoon B … +1 more , Whelan RJ
J Sep Sci · 2025 Oct · PMID 41057289 · Full text
Capillary electrophoresis (CE) is a powerful tool for studying biomolecular interactions due to its high speed, low sample consumption, and adaptability. However, challenges arise when sample buffers possess higher condu...
Capillary electrophoresis (CE) is a powerful tool for studying biomolecular interactions due to its high speed, low sample consumption, and adaptability. However, challenges arise when sample buffers possess higher conductivity than the background electrolyte (BGE), leading to peak distortions and reduced measurement accuracy in binding assays such as affinity probe CE and nonequilibrium CE of equilibrium mixtures. This study investigates these effects using a combination of simulation and experiment, focusing on aptamer-protein interactions. Moderate conductivity mismatches (e.g., sample buffer = 2 × tris glycine, BGE = 30 mM tricine) led to peak splitting artifacts, whereas large mismatches (e.g., sample buffer = phosphate-buffered saline, BGE = 30 mM tricine) produced broad, indistinct peaks, obscuring free and bound species. Simulations revealed that these artifacts arise from analyte ions trapped in high-conductivity sample plugs and are exacerbated by longer injection times. Experimental results confirmed that reducing plug length and selectively excluding artifact peaks during analysis improves quantification accuracy. When traditional separation fails under high-conductivity conditions, we propose an alternative method based on quantifying the "de-stacked" fraction of aptamers escaping the sample zone. This approach yielded values for the dissociation constant (K) and Hill coefficient (n) comparable to those obtained using fluorescence anisotropy, demonstrating its viability. The method was further validated by measuring the binding of an integrin-targeting aptamer (S10yh2) to human serum albumin. Overall, this work provides practical guidelines and analytical strategies for accurate quantification of binding interactions in CE under nonideal conductivity conditions, broadening the applicability of CE for bioanalytical research.
Preparation of In-Situ Multifunctional Silicon Self-Assembled Molecularly Imprinted Polymers for Selective Extraction of Protein.
Gong K, Wang X, Bai Y … +2 more , Ma X, Chen G
J Sep Sci · 2025 Oct · PMID 41057285 · Publisher ↗
The sol-gel method demonstrates significant potential for fabricating protein molecularly imprinted polymers (MIPs). In order to maintain good protein structure and provide more recognition functional groups during the p...
The sol-gel method demonstrates significant potential for fabricating protein molecularly imprinted polymers (MIPs). In order to maintain good protein structure and provide more recognition functional groups during the preparation of protein MIPs. In this study, an exceptionally mild sol-gel protocol for protein MIPs synthesis was developed, in which no catalyst or organic solvent was employed and abundant recognition groups were introduced through in-situ derivatization. This method has been applied in the preparation of MIPs using lysozyme (lys) as a template. The developed MIPs exhibit high adsorption capacity and selectivity toward lys. The generation of the imprinting effect was successfully demonstrated by observing the surface morphology and evaluating the physical/chemical properties of the synthesized MIPs. In subsequent adsorption experiments, MIPs also showed excellent adsorption performance (389.85 mg/g) and quickly reached the adsorption equilibrium within 60 min. In addition, the selectivity of MIPs for lys was improved by the sol-gel imprinting process, and good reusability was observed after five adsorption-desorption cycles. The method was also successfully applied to isolate lys from egg white samples. This work demonstrates the great potential of the sol-gel strategy in the field of molecular imprinting.
Development of Air-assisted Liquid-Liquid Microextraction in the Primary Amine-based Supramolecular Solvent for Determination of Tricyclic Antidepressants Prior to High-Performance Liquid Chromatography-Ultraviolet Detection.
Sabaghi M, Ezoddin M, Ghazi-Khansari M … +2 more , Javadi MS, Abdi K
J Sep Sci · 2025 Oct · PMID 41057284 · Publisher ↗
This study developed an air-assisted liquid-liquid microextraction method using a primary amine-based supramolecular solvent (SUPRAS) and a monoterpenoid as a coacervation agent to determine two tricyclic antidepressants...
This study developed an air-assisted liquid-liquid microextraction method using a primary amine-based supramolecular solvent (SUPRAS) and a monoterpenoid as a coacervation agent to determine two tricyclic antidepressants (amitriptyline and nortriptyline) from human plasma and urine samples prior to high-performance liquid chromatography analysis. The extraction of two antidepressant drugs was performed using a micellar solution of the primary amine (1-octylamine) as an amphiphile. A monoterpenoid (thymol) was utilized to initiate the coacervation process. The homogeneous solution of the SUPRAS was dispersed into the aqueous sample phase using air bubbles introduced through a syringe. The important characteristic of the suggested SUPRAS is the elimination of toxic solvents, such as tetrahydrofuran, and the removal of dispersive solvents. The experimental parameters, such as primary amine type, monoterpenoid type, extraction cycle number, and pH, were investigated. The limits of detection (signal-to-noise ratio [S/N] = 3) and quantification (S/N = 10) were achieved to be 0.5-0.6 and 1.5-1.8 µg/L with a linear range of 1.5-4000 and 1.8-4000 µg/L in urine and plasma samples. The percentage relative standard deviation values (n = 5) were 2.9%-3.5% for amitriptyline and 4.1%-4.2% for nortriptyline. The proposed method has been applied successfully to the spiked urine and plasma samples, achieving recoveries in the range of 93.1%-95.2.
Microplastic and Nanoplastic Analysis: From Pyrolysis Gas Chromatography-Mass Spectrometry to Pyrolysis Two-dimensional Gas Chromatography-Mass Spectrometry-A Critical Review.
Dumont G, Rodrigues A, Velimirovic M … +4 more , Lievens S, Jordens J, Focant JF, Stefanuto PH
J Sep Sci · 2025 Oct · PMID 41039849 · Publisher ↗
The growing environmental and health concerns regarding micro- and nanoplastics (MNPs) have prompted the development of advanced analytical methods for accurate characterization and quantification. Pyrolysis-gas chromato...
The growing environmental and health concerns regarding micro- and nanoplastics (MNPs) have prompted the development of advanced analytical methods for accurate characterization and quantification. Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) enables polymer identification by their thermal destruction into characteristic fragments. However, the small particle size and interferences originating from complex sample matrices complicate its analysis. Therefore, the integration of comprehensive two-dimensional GC (GC×GC) would improve separation efficiency and sensitivity and provide a detailed composition of environmental and biological samples. This review documents (i) the evolution of Py-GC-MS and (ii) the potential to resolve overlapping compounds, improving quantification accuracy, and detecting minor plastic compounds and degradation byproducts by comprehensive GC×GC-MS as a crucial approach to measure MNPs. Despite the documented advancements, key challenges persist. The lack of standardized protocols for sample preparation and calibration, impeding the comparability of studies, is of prime concern. The massive presence of (in)organic interferences even further accentuates the absence of internal standards in terms of quantification. Therefore, to improve analytical reliability, future research should focus on developing standardized methodologies, improving detection sensitivity for NPs, and incorporating complementary approaches. Additionally, coupling GC×GC with time-of-flight MS further strengthens its capability to provide higher analytical resolution power and better chemical description of pyrolyzates. This review highlights the crucial role of advanced Py and chromatography-based techniques in supporting the analytical description of the extent of plastic pollution and in supporting evidence-based policymaking and successful mitigation efforts to protect ecosystems and public health.
Deep Eutectic Solvents Selection for the Extraction of Phlorizin from Malus hupehensis Using Conductor-like Screening Model for Real Solvents.
Cai X, Xiong Y, Sun Q … +5 more , Wang M, Peng Y, Tang J, Li D, Liu G
J Sep Sci · 2025 Oct · PMID 41039846 · Publisher ↗
Deep eutectic solvents (DESs), composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), are efficient and widely applied solvents for extracting active compounds. Phlorizin (PHL), as the main component...
Deep eutectic solvents (DESs), composed of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs), are efficient and widely applied solvents for extracting active compounds. Phlorizin (PHL), as the main component of Malus hupehensis, plays a critical role in the preparation of a DES-based extract. The appropriate option of DES is a primary consideration in the extraction of PHL from M. hupehensis. According to the chemical structural characteristics of PHL and solvent, the conductor-like screening model for real solvents is applied to select a DES. Lactic acid (LA) and betaine (BET) with a molar ratio of 2:1 and 40 wt% water are determined, and the extraction results are consistent with the predicted solubility of PHL in selected DESs. Thus, the optimal extraction conditions are identified as liquid-material ratio 40 mL/g, extraction time 50 min, and extraction temperature 60°C, resulting in the maximum yield of 14.07 ± 0.14%. As evidenced by Fourier-transform infrared and proton nuclear magnetic resonance spectra, PHL acts as an HBD and competitively interacts with HBA sites that are occupied by LA. Molecular dynamics simulations demonstrate the hydrogen bonding interactions between PHL and DES, consisting of LA and BET, with the lowest interaction energy of -135.6 kJ/mol. Therefore, the conductor-like screening model for real solvents is an available strategy to rapidly and efficiently select the appropriate DES on the basis of the thermodynamic characteristics. DESs can effectively extract PHL from M. hupehensis, and the DES-based extract of M. hupehensis with antioxidant activity provides a foundation for further application of DESs.
A Green Method for Seized Drug Screening Using a Portable Liquid Chromatograph With UV-LED Detection.
Barber J, Lurie IS
J Sep Sci · 2025 Oct · PMID 41024379 · Publisher ↗
Portable instruments allow for on-site real-time analyses and generally take up less resources compared to standard-sized instruments. In this project, we tested the suitability of a portable liquid chromatograph for sei...
Portable instruments allow for on-site real-time analyses and generally take up less resources compared to standard-sized instruments. In this project, we tested the suitability of a portable liquid chromatograph for seized drug screening and how it, alone, may fulfill the requirements set forth by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG). For this study, we used a portable liquid chromatograph, which employed a dual column system with on-column dual UV-LED detection. All 16 drugs representing different drug classes could be identified by a combination of dual relative retention times and dual UV absorbance ratios. Most classical drugs, which represent non-novel psychoactive compounds, could be identified based on the SWGDRUG guidelines. These results suggest the potential for liquid chromatography to be performed in the field using portable devices and to provide a greener alternative to traditional liquid chromatography due to greatly reduced solvent consumption.
Determination of Lysozyme in Egg White Using Molecularly Imprinted Polymer Coated Capillary by Capillary Electrophoresis.
Gong Y, Wang Y, Zhu L … +5 more , Miao G, Fan Q, Lu B, Chen L, Wang Y
J Sep Sci · 2025 Oct · PMID 41024378 · Publisher ↗
This work aims to develop a capillary based on molecularly imprinted polymer coating for the on-line preconcentration and selective determination of lysozyme in egg white, combining the template immobilization strategy,...
This work aims to develop a capillary based on molecularly imprinted polymer coating for the on-line preconcentration and selective determination of lysozyme in egg white, combining the template immobilization strategy, surface imprinting approach, and post-imprinting modification strategy by using capillary electrophoresis. First, the capillary was coated with polydopamine and then the 3-mercaptopropionic acid coating was formed through self-assembly for subsequent immobilization of template lysozyme. Afterward, the surface-imprinted polydopamine coating was fabricated by the self-polymerization of dopamine as a functional monomer and cross-linker. After that, partially hydrolyzed poly(2-methyl-2-oxazoline), which possesses the protein-resistant adsorption ability, was introduced to reduce the nonspecific adsorption of the polydopamine coating. Finally, lysozyme molecularly imprinted polymer coated capillaries were obtained after lysozyme was eluted. The created coating was characterized by electroosmotic flow mobility measurements, scanning electron microscope, static water contact angle, and attenuated total reflection Fourier-transform infrared spectrum. Lysozyme standard solutions (concentration from 0.5 to 10.0 ng/mL) were analyzed using the fabricated capillary, achieving the detection limit of 0.1 ng/mL. In addition, the accuracy, repeatability, reproducibility, and selectivity of the prepared capillary were investigated. The recovery values of lysozyme gained were from 96.0% to 98.7% for hen egg white samples using the developed capillary.
Simultaneous Determination of Vancomycin and Creatinine by Dried Plasma Spotsampling via LC-MS/MS.
Sun C, Sun Z, Xia W … +6 more , Cao H, Qin Y, Feng Y, Ji Z, Huang J, Li Y
J Sep Sci · 2025 Oct · PMID 41024375 · Publisher ↗
Routine monitoring of vancomycin drug concentrations is crucial for optimizing dosages to ensure therapeutic efficacy and minimize adverse effects. Vancomycin's clearance is highly correlated with creatinine clearance. H...
Routine monitoring of vancomycin drug concentrations is crucial for optimizing dosages to ensure therapeutic efficacy and minimize adverse effects. Vancomycin's clearance is highly correlated with creatinine clearance. However, in developing countries, the majority of hospitals lack the on-site capacity to measure vancomycin concentrations. Dried plasma spots have emerged as an innovative alternative for biological sample collection. Dried plasma spots facilitate the convenient and rapid acquisition of plasma samples, which are highly suitable for both storage and transportation, and can be dispatched to vancomycin testing laboratories. This study presents a validated methodology for the simultaneous quantification of vancomycin and creatinine in dried plasma spots through liquid chromatography-tandem mass spectrometry. Vancomycin demonstrated excellent linearity within the range 3-50 µg/mL, while creatinine exhibited linearity from 1 to 70 µg/mL (both with r > 0.995). The trueness of all compounds was maintained within ± 15%, the precision was less than 15%, and the recoveries were within acceptable boundaries. Moreover, no significant matrix effects were detected. By employing the Passing-Bablok and Bland-Altman methods, we compared the differences and consistencies between the wet plasma concentrations and dried plasma spot concentrations of vancomycin and creatinine in 71 samples. The results revealed no significant distinctions between the two approaches and showcased comparable consistencies, implying that the dried plasma spots technique can be effectively utilized for the simultaneous determination of vancomycin and creatinine in plasma.
Identifying Oxysterols Associated With Age and Diet in Mice Using Optimized Reversed-phase Liquid Chromatography-Mass Spectrometry (RPLC-MS).
Subramaniyan I, Barr B, La-Beck NM … +3 more , Janesko BG, Gollahon L, Li L
J Sep Sci · 2025 Oct · PMID 41024374 · Full text
Structurally similar oxysterols such as 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol; 5,6α- and 5,6β-epoxycholesterol; and 24(R/S)-hydroxy cholesterol, 25-hydroxy cholesterol, and 27-hydroxycholest...
Structurally similar oxysterols such as 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol; 5,6α- and 5,6β-epoxycholesterol; and 24(R/S)-hydroxy cholesterol, 25-hydroxy cholesterol, and 27-hydroxycholesterol are traditionally difficult to resolve using reversed-phase liquid chromatography (RPLC). We present a simple yet highly optimized method for the simultaneous quantification of eight oxysterols using RPLC coupled with mass spectrometry (MS) without derivatization. Optimal separation of most oxysterols was achieved at a lower column temperature (25°C), with specific combinations of stationary and mobile phases enhancing resolution, particularly for isomeric pairs such as 7α-/7β-OHC, 5,6α-/5,6β-EC, 24 R/S-OHC, and 25-OHC. Although certain analytes (e.g., 24S-OHC and 27-OHC) remained challenging to separate due to similar retention behavior, they were distinguishable by their unique MRM transitions. We applied this method to investigate oxysterol changes in a longitudinal mouse study comparing a normal diet to a high-fat diet. Liver and brain samples were analyzed, revealing distinct distribution patterns between the two organs. Notably, 24(S)-hydroxycholesterol levels, a signature cholesterol metabolite exclusively produced in the brain, increased with age independent of diet. In contrast, 5,6α-epoxycholesterol production in the liver was influenced by both age and dietary factors. Our method provides a robust tool for studying oxysterol variation and its implications in aging and diet, offering new insights into cholesterol-derived lipid regulation across different physiological conditions.
Enhancement of GC-MS Signal for Environmental Pollutants via the Transient Matrix Effect.
Dybowski MP
J Sep Sci · 2025 Oct · PMID 41024369 · Publisher ↗
Matrix effects in gas chromatography (GC) are typically regarded as an analytical drawback, as co-eluting compounds can distort analyte quantification. This study demonstrates that a transient matrix effect can be delibe...
Matrix effects in gas chromatography (GC) are typically regarded as an analytical drawback, as co-eluting compounds can distort analyte quantification. This study demonstrates that a transient matrix effect can be deliberately induced to enhance GC-MS sensitivity for environmental pollutants. Four classes of high-boiling protectants-alcohols, amines, carboxylic acids, and polyethylene glycols (PEGs)-were systematically evaluated for their ability to increase detector response for polycyclic aromatic hydrocarbons (PAHs), chlorophenols, and nitrophenols. The influence of protectant type, sample solvent, injector temperature, and initial column temperature on signal enhancement was investigated. Among all protectants, PEGs yielded the highest improvements, with average signal increases of 280% for PAHs and 380% for chlorophenols and nitrophenols. The transient nature of the effect was confirmed through alternating injections, ensuring no cumulative influence on subsequent analyses. Application of the QuEChERS method to water and soil samples showed comparable enhancements, with minimal interference from matrix components. Method validation for PEG-400 confirmed excellent linearity (R > 0.998), two- to threefold lower LODs, and high precision (RSD < 4.3%), demonstrating robustness and reproducibility. Leveraging a controlled transient matrix effect, this approach achieves several-fold improvement in detection limits without altering chromatographic hardware, providing a simple and adaptable tool for trace-level environmental monitoring.
The Use of Single or Serially Coupled Columns and the Effect of Organic Modifier for the Separation of Constitutional Isomers of the Synthetic Cannabinoid JWH 018.
Seba JJ, Rowe WF, Lurie IS
J Sep Sci · 2025 Oct · PMID 41024357 · Publisher ↗
Traditional approaches, such as gas chromatography-mass spectrometry (GC-MS), are complemented by adopting new liquid chromatographic methods to separate isomeric drug mixtures that are unresolvable by the former techniq...
Traditional approaches, such as gas chromatography-mass spectrometry (GC-MS), are complemented by adopting new liquid chromatographic methods to separate isomeric drug mixtures that are unresolvable by the former technique. This study investigates a series of liquid chromatographic approaches using both traditional and silica hydride stationary phases. These methods focus on the separation of 11 synthetic cannabinoid constitutional isomers. Seven different columns were used to assess the utility of single and serially coupled column approaches under reversed-phase conditions. The best separation using a single column was the use of a Cogent Pentafluorophenyl (RP PFP) stationary phase, where 9 out of 11 constitutional isomers were separated. Concerning using different single columns with the same organic modifier or different organic modifiers with the same column, all compounds of interest were separated using a combination of RP PFP and RP C18 columns using methanol as the organic modifier. Cogent RP C18 separated all constitutional isomers (R = 1) in two separate runs, using methanol and acetonitrile individually. The serial coupling of columns, which did not offer better separation compared to the previous techniques, demonstrated either enhanced or diminished selectivity based on column coupling.
Determination and Confirmation of Triazine Herbicides and Their Degradation Products in Seawater by Liquid Chromatography Coupled With Quadrupole/Exactive Orbitrap Mass Spectrometry.
Sun X, Xing L, Guo M … +4 more , Jin A, Yang Y, Li Z, Yao L
J Sep Sci · 2025 Oct · PMID 41024352 · Publisher ↗
Herbicides seriously threaten the ecosystem due to their wide application and environmental migration. Nevertheless, the detection and confirmation methods of triazine herbicides and their degradation products in seawate...
Herbicides seriously threaten the ecosystem due to their wide application and environmental migration. Nevertheless, the detection and confirmation methods of triazine herbicides and their degradation products in seawater matrix remain deficient, which affect the accuracy of marine environment and aquatic organism safety risk assessment. In the present study, rapid analysis and accurate identification of seven triazine herbicides and six degradation products in seawater were achieved for the first time, based on hydrophilic-lipophilic balance solid-phase extraction combined with scanning pattern of full scan mass spectrometry-data-dependent mass spectrometry and internal standard calibration by liquid chromatography coupled with Quadrupole/Exactive Orbitrap high-resolution mass spectrometry. The results showed a good linear relationship for the tested compounds with R > 0.995. The limits of detection and quantitation of the method were 5.0 and 20 ng/L, respectively. The recoveries of the 13 compounds ranged from 70.0% to 120% when spiked at three levels, and the relative standard deviations were all less than 15% (n = 6). This method has been successfully applied to the detection of triazine herbicide in 34 real seawater samples, and the presence of prometryn, 2-hydroxy prometryn, desisopropyl prometryn, atrazine, and desisopropyl atrazine in positive seawater samples was confirmed by Quadrupole/Exactive Orbitrap high-resolution mass spectrometry. Therefore, the present method has promising application prospects in the detection and positive identification of pollutants in seawater, with high accuracy, sensitivity, and good reproducibility.
Systematic Analysis of Chemical Constituents and Pharmacokinetics of Baihe Dihuang Decoction In Vivo by UHPLC-MS/MS.
Chen Q, Xue S, Jia F … +5 more , Li M, Feng J, Jiang Y, Chen S, Fu Y
J Sep Sci · 2025 Sep · PMID 40994161 · Publisher ↗
Baihe Dihuang decoction (BDD), composed of Lilii Bulbus and Rehmanniae Radix, is a classical Chinese herbal formula. It demonstrates clinical applications in treating emotional disorders and anxiety. In this study, we ch...
Baihe Dihuang decoction (BDD), composed of Lilii Bulbus and Rehmanniae Radix, is a classical Chinese herbal formula. It demonstrates clinical applications in treating emotional disorders and anxiety. In this study, we characterized the chemical basis of BDD in vitro and elucidated its metabolic pathways, pharmacokinetic profiles, and tissue distribution in vivo. An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was used to qualitatively characterize the chemical constituents and their metabolites in rat plasma after oral administration of BDD. Then a reliable, sensitive, and accurate quantitative method based on an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to investigate the pharmacokinetics of nine major compounds and the tissue distribution of six of these compounds in rats. Results showed that 97 constituents including iridoid glycosides, phenylethanoid glycosides, phenolic acid glycerides, alkaloids, and other types of components were identified in BDD. A total of 28 prototype constituents and 66 metabolites were identified in rat plasma samples. The related metabolic pathways mainly involved deglycosylation, methylation, and deoxygenation. The pharmacokinetic results showed that the analytes displayed rapid absorption and elimination. Tissue distribution analysis revealed that all analytes were detected in heart, liver, spleen, lung, and kidney at 0.5 h after administration and presented higher concentrations in the lung and kidney compared to other tissues. This study provides a reference for clinical application and new drug development of BDD.
Simultaneous Quantification of a Novel β-Carboline Alkaloid Derivative and Its Three Metabolites in Rat Plasma by UHPLC-MS/MS and Its Application to a Pharmacokinetic Study.
Bahatijiang M, Xu N, Hu X … +11 more , Liu W, Lei J, Zhang S, Cai F, Xie Z, Zhou M, Wang J, Gao H, Teng L, Guan H, Wang C
J Sep Sci · 2025 Sep · PMID 40994126 · Publisher ↗
1-Methyl-9-(3-pyridinylmethyl)-9H-pyrido[3,4-b] indole (HMYB) is a novel semi-synthetic alkaloid derived from β-carboline harmane, which has been proven to be effective in treatment of encopresis. But the pharmacokinetic...
1-Methyl-9-(3-pyridinylmethyl)-9H-pyrido[3,4-b] indole (HMYB) is a novel semi-synthetic alkaloid derived from β-carboline harmane, which has been proven to be effective in treatment of encopresis. But the pharmacokinetic characteristics of HMYB were not yet illustrated. In this study, three metabolites (harmane, harmol, and 7-OH-HMYB) of HMYB were isolated from rat urine after oral administration of HMYB and authenticated with nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Most significantly, HMYB and its three metabolites were simultaneously quantified accurately in rat plasma using a quick, sensitive and selective UHPLC-MS/MS method. On an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm), the analytes and internal standard (osalmid) were separated by gradient elution using acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometry detector was used in positive ionization mode for multiple reaction monitoring. The method showed good linearity over the concentration range of 0.5-500 ng/mL for HMYB and three metabolites. The method was applied to the pharmacokinetic study of HMYB and its three metabolites after oral doses of HMYB (25, 50, and 100 mg/kg) and intravenous administration of 0.5 mg/kg in rats. Pharmacokinetic differences between HMYB and its three metabolites were determined, along with the absolute bioavailability of HMYB. These results would be helpful in understanding the efficacy of HMYB. The results will also contribute to providing guidance for the structural modification of more effective semi-synthetic derivatives.
Application of a Deep Learning Model to Predict Liquid Chromatography Retention Times of Food Peptides Across Chromatographic Conditions.
Hollebrands B, Hageman J, Janssen HG
J Sep Sci · 2025 Sep · PMID 40994122 · Publisher ↗
Comparing predicted and measured retention times can greatly enhance the reliability of peptide identification in LC-MS analysis of smaller, food-derived peptides where MS spectral information alone is often insufficient...
Comparing predicted and measured retention times can greatly enhance the reliability of peptide identification in LC-MS analysis of smaller, food-derived peptides where MS spectral information alone is often insufficient. Unfortunately, the extensive data sets of peptide retention times from proteomics repositories, or prediction models derived from them, have limited applicability to food-derived peptides due to the structural diversity of these peptides. To address this, we applied a transfer learning approach by fine-tuning a generic deep learning model initially trained on large proteomics datasets using our own experimental data obtained from commercial peptide standards. The method utilizes an easy to implement retraining strategy that significantly reduces data requirements and training time compared to building a model from scratch. The retrained model demonstrated strong predictive performance (Q > 0.98), and 95% of the retention time predictions of a yeast protein hydrolysate validation set fell within a ±1.0 min window across a wide range of chromatographic conditions, demonstrating both its robustness and practical relevance. We further validated this approach by applying it to the analysis of plant protein hydrolysates. The good performance seen showed its versatility and applicability for diverse sets of peptides including tryptic and non-tryptic peptides. Our work underscores the potential of transfer learning in chromatographic analysis, providing an efficient and adaptable tool for rapid and reliable peptide analysis in food research. Transfer learning enabled the utilization of extensive databases from the proteomics area in the much narrower and specialized field of food peptide analysis.
Preparation of Zwitterionic Silica-Based Sorbents for Simultaneous Extraction of Acidic and Basic Pharmaceuticals From Water Samples.
Moral A, Borrull F, Furton KG … +3 more , Kabir A, Fontanals N, Marcé RM
J Sep Sci · 2025 Sep · PMID 40984026 · Full text
The sol-gel approach was applied to develop three homemade silica sorbents. These sorbents where functionalized with mixed-mode zwitterionic exchange groups using only one additive to introduce at once the strong cation-...
The sol-gel approach was applied to develop three homemade silica sorbents. These sorbents where functionalized with mixed-mode zwitterionic exchange groups using only one additive to introduce at once the strong cation- (sulfonic groups) and strong anion-exchange (quaternary amines) moieties. The developed materials were applied for the solid-phase extraction (SPE) of basic and acidic pharmaceuticals, and the sorbent functionalized with 2-(methacryloxy)-ethyldimethyl-3-(sulfopropyl)ammonium hydroxide was the best performing one. The optimal conditions for SPE were pH 5, a variable loading volume (ranging from 25 to 100 mL depending on the complexity of the sample) and elution volume with 5 mL of 1% NHOH in MeOH. The method validation was carried out attending to apparent and relative recoveries, matrix effect, precision (intraday and interday), and detection and quantification limits. It can be highlighted that apparent recoveries were higher than 30% for most compounds and method detection limits were at the low ng/L. The validated method was applied to quantify the pharmaceuticals in environmental water samples.
Separation of Short- and Medium-Chain Fatty Acids Using Capillary Electrophoresis With Indirect Photometric Detection: Part II: Validation of Endogenous Concentration in Rat Feces.
Pham U, Sambor E, Frantz K … +1 more , Shamsi SA
J Sep Sci · 2025 Sep · PMID 40984020 · Publisher ↗
As a public health crisis, cocaine addiction has no lasting treatments to prevent relapses, and adolescent behavior has been linked to risky behavior, including drug use. Addiction treatment may benefit from targeting th...
As a public health crisis, cocaine addiction has no lasting treatments to prevent relapses, and adolescent behavior has been linked to risky behavior, including drug use. Addiction treatment may benefit from targeting the gut-brain axis. Short- and medium-chain fatty acids (SMCFAs) are produced by bacteria in the gut and communicate with the brain, therefore influencing drug reinforcement. The study has three primary objectives. To obtain dried feces, a simple, efficient desiccation time is first optimized. Second, we examined the analytical figures of merit for method validation to establish selectivity, linearity, limit of detection, limit of quantitation, precision, accuracy, matrix effects, and stability. Third, we establish baseline concentrations of SMCFAs (including branched-chain fatty acids) in the feces of healthy adults and adolescent rats. In addition, this study examines whether capillary electrophoresis with indirect photometric detection (IPD) can be used to determine whether antibiotics, cocaine, or both deplete SMCFAs in healthy rats. The results suggest that antibiotic treatment severely reduces fatty acid content in healthy rats, while cocaine exposure causes only modest decline. Therefore, the role of SMCFAs should be investigated as a possible route for gut-brain communication in addiction. To our knowledge, this is the first capillary electrophoresis IPD method that establishes baseline concentrations of fecal SMCFAs in adults and adolescents rats.
A Novel High Resolution Mass Spectrometry-Based Analytical Strategy for Simultaneous Metabolite Profiling and Standard-Free Metabolite Quantification of Artemisinin in Human Liver Microsomes and Plasma.
Du S, Liu T, Xu K … +2 more , Mao H, Xing J
J Sep Sci · 2025 Sep · PMID 40984019 · Publisher ↗
Metabolite quantification without a radiolabeled analogue or the reference standard is challenging. This study presented a novel high resolution mass spectrometry (HRMS)-based analytical strategy for simultaneous metabol...
Metabolite quantification without a radiolabeled analogue or the reference standard is challenging. This study presented a novel high resolution mass spectrometry (HRMS)-based analytical strategy for simultaneous metabolite profiling and standard-free metabolite quantification of drug candidates with limited restriction on structure. The model drug was artemisinin (ART), which is widely used in clinic to treat malaria. A major hydroxylated metabolite (M1) with minor isomer M2 was first found for ART in human liver microsomes using LC-HRMS. Second, the MS response ratio (MRR) of the hydroxylation pathway in specific biological matrix was investigated using several probe substituents (midazolam, etc.). In contrast to varying (0.5-1.9-fold difference) MS response of probe drugs and their metabolites at equimolar concentration, the MRR ratio of the hydroxylation pathway was relatively constant (∼0.6-fold). Third, simulated calculation curves for M1 were obtained based on the calibration curves of ART and the MRR ratios of the hydroxylation pathway. Fourth, the present analytical strategy provided reliable (< 31.7% deviation from that obtained using a validated LC-MS technique) quantitative data on enzyme kinetics and pharmacokinetics. As a result, M1 was found to be the predominant metabolite for ART (3.6-fold of ART exposure) in human, and another unidentified hydroxylated metabolite M2 accounted for ∼40.0% of ART exposure. The results demonstrated that the new HRMS-based analytical strategy along with the MRR ratio of a metabolic pathway evaluated by appropriate probe substituents can be a valuable tool for the simultaneous metabolite profiling and standard-free metabolite quantification in early drug development.
Introducing Hydrofluoroethers as Modifiers for Enantioseparation in Super/Subcritical Fluid Chromatography.
Farooq MQ, Miller L, Munoz L … +1 more , Roy D
J Sep Sci · 2025 Sep · PMID 40976909 · Publisher ↗
The addition of modifiers led to a marked expansion of the chemical space that can be analyzed with super/subcritical fluid chromatography. The introduction of new modifiers can help tune selectivity with commercially av...
The addition of modifiers led to a marked expansion of the chemical space that can be analyzed with super/subcritical fluid chromatography. The introduction of new modifiers can help tune selectivity with commercially available stationary phases. This work presents an entirely new class of modifiers, which offers unique selectivity not afforded by conventional solvents. We utilize hydrofluoroethers, commonly designated as Engineered Fluids, as modifier for enantioseparations on polysaccharide-based chiral stationary phases under super/subcritical fluid chromatographic conditions. Engineered fluids have been commonly utilized as heat transfer liquids for replacing chlorofluorocarbons. In this study, we utilize three different hydrofluoroethers, namely, Novec 7000, 7100, and 7200, to obtain novel selectivity on Chiralpak AD and IG columns as proof of concept for their utility. This study highlights the enhanced retention and selectivity changes afforded by these solvents, when blended with methanol in comparison to neat methanol, which is the most commonly used modifier. The effects of using different blend concentrations have also been elucidated in this study, with findings suggesting the importance of optimizing the amount of Engineered Fluid in the bulk mobile phase. The manuscript also dives into the variation in selectivity, when different hydrofluoroethers are used in the blend, pointing to the utility of each of the tested solvents.
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