First proof-of-concept of UC/HILIC for extending the versatility of the current art of supercritical fluid separation

Si-Hung, Le; Izumi, Yoshihiro; Bamba, Takeshi

doi:10.1016/j.aca.2022.340741

Cited by 6 publications

(6 citation statements)

References 51 publications

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“…Furthermore, a higher concentration of modifier at the start of the gradient favors the analyte solubility and allows higher injection volumes of polar solvents . Usually, UC methods for the analysis of metabolites employ a gradient starting from 2 to 5% of the modifier to enable the separation of lipid-related compounds, which also results in increased retention of polar metabolites. ,, Herein, there is no need to start the gradient from a low percentage of the modifier, as lipid analysis is performed through the separate injection in the first part of our SFC–MS method (see the next section). Therefore, the 20% modifier at the start of the gradient was determined as optimal for the analysis of polar metabolites.…”

Section: Resultsmentioning

confidence: 99%

“…26−29 The most recent study reported the analysis of metabolites using the extension of the UC gradient to the HILIC mode in a single measurement, which favored the elution of phosphorylated compounds. 29 However, the simultaneous analysis of polar and nonpolar metabolites in one run has limited applicability to natural samples due to the dissolution issues during the sample extraction and resuspension of the metabolite pool with an extremely wide range of physicochemical properties. In previous UC studies for the analysis of metabolites, the emphasis was mainly focused on the standards of polar compounds, while very few lipid representatives were used, apparently due to solubility reasons.…”

mentioning

confidence: 99%

“…Other attempts were focused on the application of UC to the analysis of compounds with a wide polarity range through a single injection, e.g. fat- and water-soluble vitamins, food dyes, and diverse metabolite standards. − The most recent study reported the analysis of metabolites using the extension of the UC gradient to the HILIC mode in a single measurement, which favored the elution of phosphorylated compounds . However, the simultaneous analysis of polar and nonpolar metabolites in one run has limited applicability to natural samples due to the dissolution issues during the sample extraction and resuspension of the metabolite pool with an extremely wide range of physicochemical properties.…”

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confidence: 99%

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Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography–Mass Spectrometry

Kozlov,

Hančová,

Cífková

et al. 2024

Anal. Chem.

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Supercritical fluid chromatography (SFC) is a rapidly expanding technique in the analysis of nonpolar to moderately polar substances and, more recently, also in the analysis of compounds with higher polarity. Herein, we demonstrate a proof of concept for the application of a commercial SFC instrument with electrospray ionization−mass spectrometry (MS) detection as a platform for the comprehensive analysis of metabolites with the full range of polarities, from nonpolar lipids up to highly polar metabolites. The developed single-platform SFC−MS lipidomic/metabolomic method is based on two consecutive injections of lipid and polar metabolite extracts from biphase methyl tert-butyl ether extraction using a diol column and two different gradient programs of methanol−water−ammonium formate modifier. Detailed development of the method focused mainly on the pressure limits of the system, the long-term repeatability of results, and the chromatographic performance, including optimization of the flow rate program, modifier composition and gradient, and injection solvent selection. The developed method enabled fast and comprehensive analysis of lipids and polar metabolites from plasma within a 24 min cycle with two injections using a simple analytical platform based on a single instrument, column, and mobile phase. Finally, the results from SFC−MS analysis of polar metabolites were compared with widely established liquid chromatography MS analysis in metabolomics. The comparison showed different separation selectivity of metabolites using both methods and overall lower sensitivity of the SFC−MS due to the higher flow rate and worse chromatographic performance.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography–Mass Spectrometry

Kozlov,

Hančová,

Cífková

et al. 2024

Anal. Chem.

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“…The broad polarity range accessible by applying a single gradient is highly beneficial, for example, in vitamin analysis or metabolomics studies [20,21]. In addition, the new concept of a dual gradient mode was investigated by adding a second high water content gradient through dynamic mixing of the modifier, which further increased the polarity range of capable analytes [22,23]. This now allows the elution of monomeric amino acids (AAs) and small peptides up to proteins in a single run and will assist in generating comprehensive knowledge about the impurity profile of pharmaceuticals, especially during research and development phases.…”

Section: Introductionmentioning

confidence: 99%

Controlling the elution order of insulin and its analogs in sub‐/supercritical fluid chromatography using methanesulfonic acid and 18‐crown‐6 as mobile phase additives

Neumann,

Schmidtsdorff,

Schmidt

et al. 2023

J of Separation Science

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The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed‐phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18‐crown‐6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH).

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“…In addition, Losacco et al introduced dual-gradient UC combining two gradients: the gradient of modifier in CO 2 and the gradient of water in the organic modifier . A similar approach called UC-HILIC (unified chromatography-hydrophilic interaction liquid chromatography) was published as a proof of concept for metabolomic analysis by Si-Hung in 2023 . However, none of these approaches focused on the analysis of both nonpolar small compounds, mainly analyzed by GC, and polar compounds within one run.…”

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confidence: 99%

Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts

Plachká,

Pilařová,

Kosturko

et al. 2024

Anal. Chem.

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Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS twoinjection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

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First proof-of-concept of UC/HILIC for extending the versatility of the current art of supercritical fluid separation

Cited by 6 publications

References 51 publications

Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography–Mass Spectrometry

Comprehensive Single-Platform Lipidomic/Metabolomic Analysis Using Supercritical Fluid Chromatography–Mass Spectrometry

Controlling the elution order of insulin and its analogs in sub‐/supercritical fluid chromatography using methanesulfonic acid and 18‐crown‐6 as mobile phase additives

Ultrahigh-Performance Supercritical Fluid Chromatography–Multimodal Ionization–Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts

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