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Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 μm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The applicability of ultrahigh-performance supercritical fluid chromatography coupled with mass spectrometry (UHPSFC/MS) for the qualitative analysis of metabolites with a wide polarity range (log P: -3.89-18.95) was evaluated using a representative set of 78 standards belonging to nucleosides, biogenic amines, carbohydrates, amino acids, and lipids. The effects of the gradient shape and the percentage of water (1, 2, and 5%) were investigated on the Viridis BEH column. The screening of eight stationary phases was performed for columns with different interaction sites, such as hydrogen bonding, hydrophobic, π-π, or anionic exchange type interactions. The highest number of compounds (67) of the set studied was detected on the Torus Diol column, which provided a resolution parameter of 39. The DEA column had the second best performance with 58 detected standards and the resolution parameter of 54. The overall performance of other parameters, such as selectivity, peak height, peak area, retention time stability, asymmetry factor, and mass accuracy, led to the selection of the Diol column for the final method. The comparison of additives showed that ammonium acetate gave a superior sensitivity over ammonium formate. Moreover, the influence of the ion source on the ionization efficiency was studied by employing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The results proved the complementarity of both ionization techniques, but also the superior ionization capacity of the ESI source in the negative ion mode, for which 53% of the analytes were detected compared to only 7% for the APCI source. Finally, optimized analytical conditions were applied to the analysis of a pooled human plasma sample. 44 compounds from the preselected set were detected in human plasma using ESI-UHPSFC/MS in MS E mode considering both ionization modes., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

The lipidomic research is currently devoting considerable effort to the harmonization that should enable the generation of comparable and accurate quantitative lipidomic data across different laboratories and regardless of the mass spectrometry-based platform used. In the present study, we systematically investigate the effects of the experimental setup on quantitative lipidomics data obtained by two lipid class separation approaches, hydrophilic interaction liquid chromatography (HILIC) and ultrahigh-performance supercritical fluid chromatography (UHPSFC), coupled to two different quadrupole - time of flight (QTOF) mass spectrometers from the same vendor. This approach is applied for measurements of 268 human plasma samples of healthy volunteers and renal cell carcinoma patients resulting in four data sets. We investigate and visualize differences among these data sets by multivariate data analysis methods, such as principal component analysis (PCA), orthogonal partial least square discriminant analysis (OPLS-DA), box plots, and logarithmic correlations of molar concentrations of individual lipid species. The results indicate that even measurements in the same laboratory for the same samples using different analytical platforms may yield slight variations in the molar concentrations determined. The normalization to a reference sample with defined lipid concentrations can further diminish these small differences, resulting in highly homogenous molar concentrations of individual lipid species. This strategy indicates a potential approach towards the reporting of comparable quantitative results independent from the quantitative approach and mass spectrometer used, which is important for a wider acceptance of lipidomics data in various biomarker inter-laboratory studies and ring trials., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Effects of blood collection tubes, the time period, the sample origin, and the method used on the lipidomic profile are investigated by ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS) and hydrophilic interaction liquid chromatography ultrahigh-performance liquid chromatography - mass spectrometry (HILIC-UHPLC/MS). Heparin plasma samples have been obtained from 99 healthy volunteers at three time points separated by six-month intervals together with one collection for EDTA plasma and serum. Furthermore, lipid concentrations in heparin plasma collected at two different sites are compared. 171 lipid species from eight lipid classes are quantified with UHPSFC/MS, and 122 lipid species from four lipid classes with HILIC-UHPLC/MS. The accuracy of both methods is monitored by the quantitation error using two internal standards (IS) per individual lipid classes. No significant differences in lipid profiles are observed for different time points and types of collection tubes (heparin plasma, EDTA plasma, and serum). Most pronounced lipid concentration differences are observed for the comparison of NIST SRM 1950 human plasma and mean lipid concentrations of the investigated cohort. Furthermore, differences in lipid concentrations are observed between employed methods (UHPSFC/MS vs. HILIC-UHPLC/MS), which can be compensated by the normalization using NIST SRM 1950 human plasma used as the quality control sample., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

This new analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is based on lipid class separation using 1.7 μm particle bridged ethylene hybrid silica columns and a gradient of methanol-water-ammonium acetate mixture as a modifier. The method enables a fast separation of 30 nonpolar and polar lipid classes within 6-min analysis time covering six main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive- and negative-ion full scan and tandem mass spectra measured with high mass accuracy and high resolving power. The method is used for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive, and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in clinical research.

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography - mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The potential of ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) and ultrahigh-performance supercritical fluid chromatography (UHPSFC) coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) for the analysis of 46 oxylipins and 2 fatty acid standards is compared in terms of their chromatographic resolution with the emphasis on distinguishing isobaric interferences and the method sensitivity. UHPLC provides the baseline separation of 24 isobaric oxylipins within 13min, while UHPSFC enables the separation of only 20 isobaric oxylipins within 8min. Moreover, the UHPLC/ESI-MS method provides an average improvement of sensitivity by 3.5-fold. A similar trend is observed in the analysis of human plasma samples, but lower ion suppression effects caused by lysophospholipids (LPL) are observed in case of UHPSFC/ESI-MS due to better separation of LPL. Both methods are fully applicable for the analysis of oxylipins, but UHPLC/ESI-MS method is preferred due to better separation and higher sensitivity, which results in the identification of 31 oxylipins in human plasma based on available standards and additional tentative 20 identifications based on accurate m/z values and the fragmentation behavior known from the literature., (Copyright © 2017 Elsevier B.V. All rights reserved.)

New analytical approach for high-throughput and comprehensive lipidomic analysis of biological samples using ultrahigh-performance supercritical fluid chromatography (UHPSFC) with electrospray ionization-mass spectrometry (ESI-MS) is presented in this work as an alternative approach to established shotgun MS or high-performance liquid chromatography-MS. The lipid class separation is performed by UHPSFC method based on 1.7 μm particle-bridged ethylene hybrid silica column with a gradient of methanol-water-ammonium acetate mixture as a modifier. All parameters of UHPSFC conditions are carefully optimized and their influence on the chromatographic behavior of lipids is discussed. The final UHPSFC/ESI-MS method enables a fast separation of 30 nonpolar and polar lipid classes within 6 min analysis covering 6 main lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. Individual lipid species within lipid classes are identified based on positive and negative-ion full-scan and tandem mass spectra measured with high mass accuracy and high resolving power. Developed UHPSFC/ESI-MS method is applied for the analysis of porcine brain extract as a complex lipidomic sample, where 24 lipid classes containing 436 lipid species are identified. The method is validated for the quantitative analysis of lipid species in biological tissues using internal standards for each lipid class. This high-throughput, comprehensive and accurate UHPSFC/ESI-MS method is suitable for the lipidomic analysis of large sample sets in the clinical research.