Journal of separation science最新文献

Metabolomics, a powerful discipline within systems biology, aims at comprehensive profiling of small molecules in biological samples. The challenges of biological sample complexity are addressed through innovative sample preparation methods, including solid-phase extraction and microextraction techniques, enhancing the detection and quantification of low-abundance metabolites. Advances in chromatographic separation, particularly liquid chromatography (LC) and gas chromatography (GC), coupled with high-resolution (HR) mass spectrometry (MS), have significantly improved the sensitivity, selectivity, and throughput of metabolomic studies. Cutting-edge techniques, such as ion-mobility mass spectrometry (IM-MS) and tandem MS (MS/MS), further expand the capacity for comprehensive metabolite profiling. These advanced analytical platforms each offer unique advantages for metabolomics, with continued technological improvements driving deeper insights into metabolic pathways and biomarker discovery. By providing a detailed overview of current trends and techniques, this review aims to offer valuable insights into the future of metabolomics in human health research and its translational potential in clinical settings. Toward the end, this review also highlights the biomedical applications of metabolomics, emphasizing its role in biomarker discovery, disease diagnostics, personalized medicine, and drug development.

Favelines, benzocycloheptene diterpenes exclusively found in Cnidoscolus quercifolius, have shown promising anticancer properties, particularly phyllacanthone. Nevertheless, the isolation of these compounds remains challenging due to their similar polarity. The use of centrifugal partition chromatography (CPC) has enabled the quick and efficient purification of phyllacanthone from C. quercifolius crude extract with a yield of 4.5% (w/w) and a purity of 83%. Favelines-enriched fractions were also obtained and purified by preparative HPLC, resulting in the isolation of faveline, isofavelol, and deoxofaveline. In addition, phyllacanthone B, a previously unreported derivative, was identified and characterized. Pharmacological evaluations revealed the significant anti-melanoma activity of purified metabolites, with distinct structure–activity relationships. These results shed light on the mechanism of action of favelines, suggesting their potential as hits for the development of novel anticancer agents.

Favelines, benzocycloheptene diterpenes exclusively found in Cnidoscolus quercifolius, have shown promising anticancer properties, particularly phyllacanthone. Nevertheless, the isolation of these compounds remains challenging due to their similar polarity. The use of centrifugal partition chromatography (CPC) has enabled the quick and efficient purification of phyllacanthone from C. quercifolius crude extract with a yield of 4.5% (w/w) and a purity of 83%. Favelines-enriched fractions were also obtained and purified by preparative HPLC, resulting in the isolation of faveline, isofavelol, and deoxofaveline. In addition, phyllacanthone B, a previously unreported derivative, was identified and characterized. Pharmacological evaluations revealed the significant anti-melanoma activity of purified metabolites, with distinct structure–activity relationships. These results shed light on the mechanism of action of favelines, suggesting their potential as hits for the development of novel anticancer agents.

Exosomes, a subset of extracellular vesicles (EVs) ranging in size from 30 to 150 nm, are of significant interest for biomedical applications such as diagnostic testing and therapeutics delivery. Biofluids, including urine, blood, and saliva, contain exosomes that carry biomarkers reflective of their host cells. However, isolation of EVs is often a challenge due to their size range, low density, and high hydrophobicity. Isolations can involve long separation times (ultracentrifugation) or result in impure eluates (size exclusion chromatography, polymer-based precipitation). As an alternative to these methods, this study evaluates the first use of nylon-6 capillary-channeled polymer (C-CP) fiber columns to separate EVs from human urine via a step-gradient hydrophobic interaction chromatography method. Different from previous efforts using polyester fiber columns for EV separations, nylon-6 shows potential for increased isolation efficiency, including somewhat higher column loading capacity and more gentle EV elution solvent strength. The efficacy of this approach to EV separation has been determined by scanning electron and transmission microscopy, nanoparticle flow cytometry (NanoFCM), and Bradford protein assays. Electron microscopy showed isolated vesicles of the expected morphology. Nanoparticle flow cytometry determined particle densities of eluates yielding up to 5 × 10⁸ particles mL⁻¹, a typical distribution of vesicle sizes in the eluate (60–100 nm), and immunoconfirmation using fluorescent anti-CD81 antibodies. Bradford assays confirmed that protein concentrations in the EV eluate were significantly reduced (approx. sevenfold) from raw urine. Overall, this approach provides a low-cost and time-efficient (< 20 min) column separation to yield urinary EVs of the high purities required for downstream applications, including diagnostic testing and therapeutics.

Exosomes, a subset of extracellular vesicles (EVs) ranging in size from 30 to 150 nm, are of significant interest for biomedical applications such as diagnostic testing and therapeutics delivery. Biofluids, including urine, blood, and saliva, contain exosomes that carry biomarkers reflective of their host cells. However, isolation of EVs is often a challenge due to their size range, low density, and high hydrophobicity. Isolations can involve long separation times (ultracentrifugation) or result in impure eluates (size exclusion chromatography, polymer-based precipitation). As an alternative to these methods, this study evaluates the first use of nylon-6 capillary-channeled polymer (C-CP) fiber columns to separate EVs from human urine via a step-gradient hydrophobic interaction chromatography method. Different from previous efforts using polyester fiber columns for EV separations, nylon-6 shows potential for increased isolation efficiency, including somewhat higher column loading capacity and more gentle EV elution solvent strength. The efficacy of this approach to EV separation has been determined by scanning electron and transmission microscopy, nanoparticle flow cytometry (NanoFCM), and Bradford protein assays. Electron microscopy showed isolated vesicles of the expected morphology. Nanoparticle flow cytometry determined particle densities of eluates yielding up to 5 × 10⁸ particles mL⁻¹, a typical distribution of vesicle sizes in the eluate (60–100 nm), and immunoconfirmation using fluorescent anti-CD81 antibodies. Bradford assays confirmed that protein concentrations in the EV eluate were significantly reduced (approx. sevenfold) from raw urine. Overall, this approach provides a low-cost and time-efficient (< 20 min) column separation to yield urinary EVs of the high purities required for downstream applications, including diagnostic testing and therapeutics.

Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 µm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 µm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50–15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.

Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 µm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 µm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50–15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.