Deuterated Modifiers in Sub/Supercritical Fluid Chromatography for Streamlined NMR Structure Elucidation.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO2) and deuterated modifiers (CD3OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH3OH) and its deuterated counterpart (CD3OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

In Silico Method Development of Achiral and Chiral Tandem Column Reversed-phase Liquid Chromatography for Multicomponent Pharmaceutical Mixtures.

Tandem column liquid chromatography (LC) is a convenient, cost-effective approach to resolve multicomponent mixtures by serially coupling columns on readily available one-dimensional separation systems without specialized user training. Yet, adoption of this technique remains limited, mainly due to the difficulty in identifying optimal selectivity out of many possible tandem column combinations. At this point, method development and optimization require laborious "hit-or-miss" experimentation and "blind" screening when investigating different column selectivity without standard analytes. As a result, many chromatography practitioners end up combining two columns of similar selectivity, limiting the scope and potential of tandem column LC as a mainstay for industrial applications. To circumvent this challenge, we herein introduce a straightforward in silico multifactorial approach as a framework to expediently map the separation landscape across multiple tandem columns (achiral and chiral) and eluent combinations (isocratic and gradient elution) under reversed-phase LC conditions. Retention models were built using commercially available LC simulator software showcasing less than 2% difference between experimental and simulated retention times for analytes of interest in multicomponent pharmaceutical mixtures (e.g., metabolites and cyclic peptides).

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Enantioselective UHPLC Screening Combined with In Silico Modeling for Streamlined Development of Ultrafast Enantiopurity Assays.

Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 µm fully porous and sub-3 µm superficially porous particles) with nine mobile phase eluent choices. This automated setup operates under a vast selection of reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, polar-organic mode, and polar-ionic mode conditions with minimal manual intervention and high success rate. Examples of highly efficient enantioseparations are illustrated from the integration of chiral screening conditions and computer-assisted modeling. Furthermore, we describe the nuances of in silico method development for chiral separations via second-degree polynomial regression fit using LC simulator (ACD/Labs) software. The retention models were found to be very accurate for chiral resolution of single and multicomponent mixtures of enantiomeric species across different types of CSPs, with differences between experimental and simulated retention times of less than 0.5%. Finally, we illustrate how this approach lays the foundation for a streamlined development of ultrafast enantioseparations applied to high-throughput enantiopurity analysis and its use in the second dimension of two-dimensional liquid chromatography experiments.

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals.

The mounting complexity of new modalities in the biopharmaceutical industry entails a commensurate level of analytical innovations to enable the rapid discovery and development of novel therapeutics and vaccines. Hydrophobic interaction chromatography (HIC) has become one of the widely preferred separation techniques for the analysis and purification of biopharmaceuticals under nondenaturing conditions. Inarguably, HIC method development remains very challenging and labor-intensive owing to the numerous factors that are typically optimized by a "hit-or-miss" strategy (e.g., the nature of the salt, stationary phase chemistry, temperature, mobile phase additive, and ionic strength). Herein, we introduce a new HIC method development framework composed of a fully automated multicolumn and multieluent platform coupled with in silico multifactorial simulation and integrated fraction collection for streamlined method screening, optimization, and analytical-scale purification of biopharmaceutical targets. The power and versatility of this workflow are showcased by a wide range of applications including trivial proteins, monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), oxidation variants, and denatured proteins. We also illustrate convenient and rapid HIC method development outcomes from the effective combination of this screening setup with computer-assisted simulations. HIC retention models were built using readily available LC simulator software outlining less than a 5% difference between experimental and simulated retention times with a correlation coefficient of >0.99 for pharmaceutically relevant multicomponent mixtures. In addition, we demonstrate how this approach paves the path for a straightforward identification of first-dimension HIC conditions that are combined with mass spectrometry (MS)-friendly reversed-phase liquid chromatography (RPLC) detection in the second dimension (heart-cutting two-dimensional (2D)-HIC-RPLC-diode array detector (DAD)-MS), enabling the analysis and purification of biopharmaceutical targets.

Automated ion exchange chromatography screening combined with in silico multifactorial simulation for efficient method development and purification of biopharmaceutical targets.

Bioprocess development of increasingly challenging therapeutics and vaccines requires a commensurate level of analytical innovation to deliver critical assays across functional areas. Chromatography hyphenated to numerous choices of detection has undeniably been the preferred analytical tool in the pharmaceutical industry for decades to analyze and isolate targets (e.g., APIs, intermediates, and byproducts) from multicomponent mixtures. Among many techniques, ion exchange chromatography (IEX) is widely used for the analysis and purification of biopharmaceuticals due to its unique selectivity that delivers distinctive chromatographic profiles compared to other separation modes (e.g., RPLC, HILIC, and SFC) without denaturing protein targets upon isolation process. However, IEX method development is still considered one of the most challenging and laborious approaches due to the many variables involved such as elution mechanism (via salt, pH, or salt-mediated-pH gradients), stationary phase's properties (positively or negatively charged; strong or weak ion exchanger), buffer type and ionic strength as well as pH choices. Herein, we introduce a new framework consisting of a multicolumn IEX screening in conjunction with computer-assisted simulation for efficient method development and purification of biopharmaceuticals. The screening component integrates a total of 12 different columns and 24 mobile phases that are sequentially operated in a straightforward automated fashion for both cation and anion exchange modes (CEX and AEX, respectively). Optimal and robust operating conditions are achieved via computer-assisted simulation using readily available software (ACD Laboratories/LC Simulator), showcasing differences between experimental and simulated retention times of less than 0.5%. In addition, automated fraction collection is also incorporated into this framework, illustrating the practicality and ease of use in the context of separation, analysis, and purification of nucleotides, peptides, and proteins. Finally, we provide examples of the use of this IEX screening as a framework to identify efficient first dimension (1D) conditions that are combined with MS-friendly RPLC conditions in the second dimension (2D) for two-dimensional liquid chromatography experiments enabling purity analysis and identification of pharmaceutical targets.

Dual-Gradient Unified Chromatography: A New Paradigm for Versatility in Simultaneous Multicomponent Analysis.

Generality in analytical chemistry can be manifested in impactful platforms that can streamline modern organic synthesis and biopharmaceutical processes. We herein introduce a hybrid separation technique named Dual-Gradient Unified Chromatography (DGUC), which is built upon an automated dynamic modulation of CO2 , organic modifier, and water blends with various buffers. This concept enables simultaneous multicomponent analysis of both small and large molecules across a wide polarity range in single experimental runs. After a careful investigation of its fundamental aspects, a DGUC-DAD-MS screening workflow that combines multiple orthogonal column and mobile phase choices across a far-reaching universal elution profile is also reported. The power of this framework is demonstrated with new analytical applications guiding academic and industrial laboratories in the development of new (bio)pharmaceutical targets (e.g. synthetic intermediates, nucleosides, cyclic and linear peptides, proteins, antibody drug conjugates).

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3) a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Unified chromatography in drug development: Exploiting chaotropic/kosmotropic salts for an accelerated method development.

Recent trends in supercritical fluid chromatography (SFC) introduced an innovative gradient profile called Unified Chromatography (UC), which pushes the amount of liquid modifier up to 80-100 % of the total mobile phase composition. These new conditions allow the full transition from a supercritical to a liquid state, unifying the benefits of both SFC and liquid chromatography. However, to facilitate the use of UC for industrial drug development, a stronger effort is needed to streamline and simplify its method development and optimization. In this work, a quick and novel method development procedure for UC is introduced, enabled by the first-time use of novel additives in SFC/UC that exploit chaotropic/kosmotropic properties. A comprehensive view on some fundamental properties, such as the amount of liquid modifier blended with supercritical CO2 (scCO2) and the percentage of water added in the mobile phase is given, to clarify the benefits of using either a chaotropic salt (NaClO4), kosmotropic (HCOONa) or salt with mixed properties (NaOMs - sodium methanesulfonate). With this expanded knowledge, challenging separations of nucleosides, nucleotide, indoles, triazoles and related derivates have been accomplished with UC. Finally, we provide an example of UC delivering a faster and better method for an AbbVie pipeline compound under accelerated stability study. The combined use of scCO2-based chromatography and the novel additive NaClO4 ensures the retention and elution of all degradation species generated at different conditions, where RP-HPLC failed to provide satisfactory performance.

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.

Parallel chiral sub/supercritical fluid chromatography screening as a framework for accelerated purification of pharmaceutical targets.

Chiral sub/supercritical fluid chromatography (SFC) has established itself as one of the preferred techniques for enantioseparations at both analytical and preparative scale. Herein, we introduce a parallel multicolumn SFC screening for automated chiral method development in fast-paced settings. The practicality and speed advantages of this approach are illustrated with parallel screening of a diverse set of chiral molecules across ten columns with five different organic modifiers/CO2 based eluents enabling rapid identification of suitable enantioseparation conditions for accelerated purification of pharmaceutical targets. Rapid delivery turnarounds of pure enantiomers of less than 1 h from screening to target isolation are demonstrated illustrating the power of this approach.

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

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.

Ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry for antidoping analyses: Assessment of the inter-laboratory reproducibility with urine samples.

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses. To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven "blind" urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.

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.

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%).

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. I - Optimization of separation conditions for the simultaneous analysis of hydrophilic and lipophilic substances.

The aim of this study was to evaluate the suitability of SFC-MS for the analysis of a wide range of compounds including lipophilic and highly hydrophilic substances (log P values comprised between -6 and 11), for its potential application toward human metabolomics. For this purpose, a generic unified chromatography gradient from 2 to 100% organic modifier in CO2 was systematically applied. In terms of chemistry, the best stationary phases for this application were found to be the Agilent Poroshell HILIC (bare silica) and Macherey-Nagel Nucleoshell HILIC (silica bonded with a zwitterionic ligand). To avoid system overpressure at very high organic modifier proportion, columns of 100 × 3 mm I.D. packed with sub-3 µm superficially porous particles were selected. In terms of organic modifier, a mixture of 95% MeOH and 5% water was selected, with 50 mM ammonium formate and 1 mM ammonium fluoride, to afford good solubility of analytes in the mobile phase, limited retention for the most hydrophilic metabolites and suitable peak shapes of ionizable species. A sample diluent containing 50%ACN/50% water was employed as injection solvent. These conditions were applied to a representative set of metabolites belonging to nucleosides, nucleotides, small organic acids, small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances, quaternary ammonium metabolites, phosphate-based substances, carbohydrates and amino acids. Among all these metabolites, 65% of the compounds were adequately analyzed with excellent peak shape, 23% provided distorted peak shapes, while only 12% were not detected (mostly metabolites having several phosphate or several carboxylic acid groups).