Evolution of sub/supercritical fluid chromatography – mass spectrometry for the analysis of highly polar compounds and biological matrices

The scope of this thesis has focused on the investigation of the performance and robustness of UHPSFC-MS in routine laboratories. Furthermore, the analysis of highly polar compounds was tested using UHPSFC, to verify the potential attractiveness of this technique not only as an alternative to RPLC but also to HILIC analysis, in which strongly polar and hydrophilic substances are analyzed. To this purpose, UHPSFC has been applied in different fields, such as untargeted metabolomics employing different biological matrices and peptide analysis. Finally, some fundamental aspects of UHPSFC have been also the subject of work, in an attempt to complement the knowledge on the mechanisms controlling this technique

Metamorphosis of supercritical fluid chromatography: A viable tool for the analysis of polar compounds?

Recent developments in supercritical fluid chromatography (SFC) have highlighted the applicability of this technique for the analysis of highly polar compounds. The combination of polar stationary phases and CO2-based mobile phases with an increasing presence of liquid co-solvent (up to 100%) has enabled to further expand the application field of SFC towards a variety of samples such as polar endogenous metabolites, plant extracts, water-soluble vitamins, pesticides, sugars, peptides and so on. In this evolution, a key role was played by the addition of up to 5e10% of water in the liquid co-solvent. Moreover, the presence of water enabled higher concentrations of additives, up to 75e100 mM in some cases. These improved conditions were fundamental in expanding the applicability range of SFC. Overall, SFC has demonstrated its evolution into a mature technique capable of offering a true alternative to liquid chromatography for the analysis of polar compounds

Supercritical fluid chromatography – Mass spectrometry: Recent evolution and current trends

Supercritical fluid chromatography (SFC) has recently experienced renovated impulse from research groups. Its hyphenation to mass spectrometers (MS) proved to be of significant importance in catalysing interest from researchers. In contrast to liquid chromatography (LC), the coupling of SFC-MS requires the use of an interface in order to deal efficiently with the decompression of supercritical CO2 and possible precipitation issues of samples while entering the ionization chamber. The most common SFC-MS interfaces employ an additional sheath pump that reduces sample precipitation. However, there are still issues in dealing with the CO2 decompression phenomenon, with different solutions being given. Matrix effects (MEs) under SFC-MS have proved to be quite different from those generally observed in LC-MS, with ion suppression being the main form of ME. Nonetheless, SFC-MS is capable of reaching comparable sensitivity values to LC-MS, and in some cases performing even better. Several applications have been recently developed for SFC-MS, spacing from the analysis of plant extracts, biological matrices for anti-doping and forensic purposes, as well as highly polar compounds such as carbohydrates and endogenous metabolites

Application space for SFC in pharmaceutical drug discovery and development

editorial reviewedAbstract The purpose of this chapter is to provide a brief overview of the past, present, and future applications of supercritical fluid chromatography (SFC) in pharmaceutical drug discovery and development. In the first part of this chapter, various examples are provided, showing how SFC has been used in the past. Originally employed for the analysis of apolar compounds, SFC rapidly became an interesting tool to substitute NPLC for chiral separations, due to its higher efficiency, faster analysis times, reduced environmental impact, and lower costs. Developments in the early 2000s begun exploring the usefulness of SFC also for achiral analyses, but the instrumentation available was not capable to meet the well-established robustness and sensitivity criteria used in liquid and gas chromatography, thus discouraging its implementation in pharmaceutical analysis. However, the introduction of ultra-high-performance supercritical fluid chromatography (UHPSFC) systems from 2012 has played a key role in refueling interest in this technique. With better instrumentation, UHPSFC demonstrated very promising results in the pharmaceutical industry also in the context of achiral separations, from the impurity profiling of synthetic APIs to its implementation in more challenging contexts, such as peptide analysis. In the second part of the chapter, considerations on the SFC stationary and mobile phases, its coupling to detectors (especially MS), and performance in the context of method validation are discussed. The recent evolution of SFC has depicted an image of this technique being efficient, fast, complementary to liquid chromatography (LC), and able to satisfy different validation criteria. Finally, an interesting perspective for SFC is linked to its implementation in multidimensional systems, with the aim to expand chromatographic selectivity

Application space for SFC in pharmaceutical drug discovery and development

editorial reviewedAbstract The purpose of this chapter is to provide a brief overview of the past, present, and future applications of supercritical fluid chromatography (SFC) in pharmaceutical drug discovery and development. In the first part of this chapter, various examples are provided, showing how SFC has been used in the past. Originally employed for the analysis of apolar compounds, SFC rapidly became an interesting tool to substitute NPLC for chiral separations, due to its higher efficiency, faster analysis times, reduced environmental impact, and lower costs. Developments in the early 2000s begun exploring the usefulness of SFC also for achiral analyses, but the instrumentation available was not capable to meet the well-established robustness and sensitivity criteria used in liquid and gas chromatography, thus discouraging its implementation in pharmaceutical analysis. However, the introduction of ultra-high-performance supercritical fluid chromatography (UHPSFC) systems from 2012 has played a key role in refueling interest in this technique. With better instrumentation, UHPSFC demonstrated very promising results in the pharmaceutical industry also in the context of achiral separations, from the impurity profiling of synthetic APIs to its implementation in more challenging contexts, such as peptide analysis. In the second part of the chapter, considerations on the SFC stationary and mobile phases, its coupling to detectors (especially MS), and performance in the context of method validation are discussed. The recent evolution of SFC has depicted an image of this technique being efficient, fast, complementary to liquid chromatography (LC), and able to satisfy different validation criteria. Finally, an interesting perspective for SFC is linked to its implementation in multidimensional systems, with the aim to expand chromatographic selectivity

Investigating the use of unconventional temperatures in supercritical fluid chromatography

The use of unorthodox temperatures, ranging from −5 °C up to 80 °C, have been thoroughly investigated in supercritical fluid chromatography. To this purpose, an initial evaluation of the kinetic and thermodynamic performance has been made with a set of 4 analytes eluting at different percentages of organic co-solvent in the mobile phase (3%–10% - 45%–80%). The van Deemter plots have demonstrated how, at low organic modifier presence, the use of low temperatures did not necessarily translate into worse performance, while high temperatures could pose more issues due to the poor handling of the super/subcritical mobile phase by the chromatographic system. With important percentages of co-solvent, however, high temperatures were fundamental in ensuring better profiles of the van Deemter plots, compared to low temperatures. Pressure plots have demonstrated that gradients reaching elevated percentages of organic modifiers can also be used on stationary phases packed with sub 2 μm silica particles if high temperatures are employed. The thermodynamic evaluation, made via the analysis of van’t Hoff plots, indicates the presence of three retention behaviors happening in UHPSFC when switching from high to low temperatures, depending on the co-solvent percentage needed to elute one analyte. Finally, an assessment of the stationary phase stability at high temperatures was performed: the retention times variabilities recorded were minimal (RSD < 2.5%), as well as the peak widths and inlet column pressures were somewhat constant throughout the analyses. In the second part of this study, a focus on potential applications benefiting from such unconventional temperatures has been made. A series of challenging analytes have experienced better chromatographic resolution at either high or low temperatures, providing therefore a potentially interesting tool to analysts during the chromatographic method development process. In conclusion, the UV sensitivity at different temperatures was also taken into consideration, with no significant impact on the quality of the UV signal under any condition

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL-1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL-1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.</p

Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min

Supercritical fluid chromatography–mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC2 Torus 2-Picolylamine (2-PIC), UPC2 Viridis BEH and Acquity UPLC HSS C18 SB. Four columns, per column chemistry, have been purchased to represent three different production lots, with a total of twelve columns employed in this study. The two columns from the same lot were applied to the first part of the study (repeatability), whereas the representative of three different lots were employed in the second part (robustness). In terms of organic modifier, a mixture of 98% MeOH and 2% water containing 20 mM ammonium formate was selected in order to limit the formation of methyl-silyl ethers on the surface of the silica particles, thus potentially improving the repeatability of retention times. A comparison with an UHPLC reference analytical method was made with the same set of analytes. The average relative standard deviations (RSD%), represented in split violin plots, illustrate how two of the UHPSFC columns assessed in this study were able to generate an excellent repeatability of retention times, with results that are in a similar range of those generated by UHPLC. Moreover, the Torus 2-PIC has proven to be the best of the three stationary phases, with an impressive RSD% of 0.5% in diluted urine relative to the inter-month variability. Finally, the inter-batch reproducibility assessment has highlighted a good reproducibility of the same stationary phase belonging to different production lots for all three column chemistries assessed, with the Viridis BEH silica generating an RSD% of 0.7% in diluted urine. Higher values of RSD (%) were found for Torus 2-PIC and HSS C18 SB, respectively of 1.0% and 1.6%

Applicability of Supercritical fluid chromatography–Mass spectrometry to metabolomics. II–Assessment of a comprehensive library of metabolites and evaluation of biological matrices

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks. In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%)