Analysis of free amino acids with unified chromatography-mass spectrometry-application to food supplements.

Amino acids are most often analyzed in reversed-phase liquid chromatography after a derivatization procedure to render them sufficiently hydrophobic and detectable with UV or fluorimetric detection. Simpler methods should be possible to avoid additional chemical reactions. We present an improved method to analyze free amino acids with unified chromatography, that is to say with a wide elution gradient starting with supercritical fluid chromatography (SFC) conditions (high percentage of carbon dioxide) and ending with high-performance liquid chromatography (HPLC) conditions (100% co-solvent). The mobile phase composition was carefully adjusted to permit the elution of 21 natural amino acids (among which 19 proteinogenic) with very good peak shapes from a zwitterionic cinchona-based stationary phase (Chiralpak ZWIX(+)). Chiral separation was not desired. The mobile phase finally selected comprised carbon dioxide and a co-solvent (methanol containing 2% water and 20 mM methanesulfonic acid), ranging from 10 to 100% in 7 min followed by 3 min re-equilibration at 25 °C. A reversed pressure gradient (15 to 11 MPa) and a reversed flow rate gradient (3 to 1 mL/min) were applied to avoid reaching the upper pressure limit of the pumping system (40 MPa) and to favor high chromatographic efficiency at every stage of the elution gradient. Detection was achieved with electrospray ionization-mass spectrometry (ESI(+)-MS). The method is then fast and straightforward as no derivatization step is necessary, and all isobaric species were chromatographically resolved. To demonstrate the applicability of the method, it was applied to the quantitation of amino acids in food supplements commonly consumed by sportsmen, containing taurine (a common natural amino acid) or branched-chain amino acids (BCAA), namely valine, and the isobaric leucine and isoleucine. A standard addition method was examined for sensitivity, linearity, repeatability and intermediate precision.

Cinchona-based zwitterionic stationary phases: Exploring retention and enantioseparation mechanisms in supercritical fluid chromatography with a fragmentation approach.

Chiralpak ZWIX(+) and ZWIX(-), are brush-type bonded-silica chiral stationary phases (CSPs), based on complex diastereomeric Cinchona alkaloids derivatives bearing both a positive and a negative charge. In the present study, we aimed to improve the understanding of retention and enantioseparation mechanisms of these CSPs employed in supercritical fluid chromatography (SFC). For this purpose, 9 other stationary phases were used as comparison systems: two of them are commercially available and bear only a positive charge (Chiralpak QN-AX and QD-AX) and the 7 others were designed purposely to be structurally similar to the parent ZWIX phases, but miss some portion of the complex ligand. First, cluster analysis was employed to identify similar and dissimilar behavior among the 11 stationary phases, where ionic interactions appeared to dominate the observed differences. Secondly, the stationary phases were characterized with linear solvation energy relationships (LSER) based on the SFC analysis of 161 achiral analytes and a modified version of the solvation parameter model to include ionic interactions. This served to compare the interaction capabilities for the 11 stationary phases and showed in particular the contribution of attractive and repulsive ionic interactions. Then the ZWIX phases were characterized for their enantioseparation capabilities with a set of 58 racemic probes. Discriminant analysis was applied to explore the molecular structural features that are useful to successful enantioseparation on the ZWIX phases. In particular, it appeared that the presence of positive charges in the analyte is causing increased retention but is not necessarily a favorable feature to enantiorecognition. On the opposite, the presence of negative charges in the analyte favors early elution and enantiorecognition. Finally, a smaller set of 30 pairs of enantiomers, selected by their structural diversity and different enantioseparation values on the ZWIX phases, were analyzed on all chiral phases to observe the contribution of each structural fragment of the chiral ligand on enantioselectivity. Molecular modelling of the ligands also helped in understanding the three-dimensional arrangement of each ligand, notably the intra-molecular hydrogen bonding or the possible contribution of ionic interactions. In the end, each structural element in the ZWIX phases appeared to be a significant contributor to successful enantioresolution, whether they contribute as direct interaction groups (ion-exchange functions) or as steric constraints to orientate the interacting groups towards the analytes.

Effects of high concentrations of mobile phase additives on retention and separation mechanisms on a teicoplanin aglycone stationary phase in supercritical fluid chromatography.

The objective of this study is to understand the behavior of a peptide in a medium containing supercritical carbon dioxide mixed with an alcohol (methanol) and acidic or basic additives in uncommonly high concentrations. Chirobiotic TAG is a chromatographic column made of silica bonded with a macrocyclic peptide, teicoplanin aglycone. With this stationary phase, two additives (trifluoroacetic acid and isopropylamine) were tested under extreme concentration conditions to observe the behavior of this peptide. Indeed, concentrations exceeding 1 M in the methanol co-solvent (>0.1 M overall concentration in the CO2-methanol mixture) were used whereas usual additive concentrations employed in supercritical fluid chromatography (SFC) rarely exceed 50 mM in the co-solvent. One purpose was to modify the apparent pH of the fluid, which is normally slightly acidic (around 5) and consequently possibly changing the ionization state of the peptide. Firstly, the effect of acidic and basic additives on the polarity and the apparent pH were evaluated with the help of color indicators. This served to assess the ionization state of the peptide under the selected operating conditions. Secondly, 54 achiral and 24 chiral molecules were injected in the chromatographic column at different levels of additives. The achiral species served at establishing retention models based on linear solvation energy relationships (LSER), while the chiral species were examined for their enantioresolution. From the LSER equations and observation of chromatograms, it appeared that specific interactions between the peptide-based stationary phase and the analytes evolved when increasing the concentration of additives, particularly hydrogen bonds and ionic interactions. A bare silica stationary phase (Acquity BEH) served as reference to deconvolute the contributions of silica support and bonded peptide. This study, with these extreme conditions of mobile phase, could be useful to understand the behavior of such peptides in SFC mobile phases and also improve the knowledge of the effects of additives in SFC, which should be helpful in the future prospect of analyzing large biomolecules in SFC.

A chiral unified chromatography-mass spectrometry method to analyze free amino acids.

In this project, we aimed at analyzing native (or free) amino acids with supercritical fluid chromatography coupled to mass spectrometric detection, with modern instruments and methods, and maintaining as simple a mobile phase as possible to ensure applicability of the method. The purpose was twofold: (i) a generic method allowing for satisfactory elution of a wide range of amino acids (acidic, basic, or neutral residue) and (ii) resolution of the enantiomeric pairs. The Chiralpak ZWIX (+) and (-) stationary phases were selected as they are well-known for the enantioresolution of amino acids in liquid chromatographic modes. A wide range elution gradient, starting with a large concentration of carbon dioxide (90%) and finishing at 100% solvent (methanol containing 70 mM ammonium formate and 7% water) allowed the elution of 18 native proteinogenic amino acids out of 19 injected. In these conditions, enantioselectivity was achieved for 16 of them. The basic amino acids (arginine, histidine, and lysine) were the most difficult to elute in these conditions, resulting in rather poor peak shapes. Cysteine was never observed in any of the conditions tested. Sample application was attempted with two food supplements, tablets containing a mixture of 17 proteinogenic amino acids and capsules containing taurine and theanine that were not present in the standards used for the method development. The sample preparation method was very simple, involving dissolution of the tablets and capsules in acidified water, filtration, and dilution with methanol. Mass spectrometric detection (electrospray ionization with single-quadrupole mass detection) allowed for unambiguous identification of most amino acids, except for the leucine and isoleucine isomers that were not separated by the generic gradient. The observation of taurine and theanine also suggests that the method should be generally applicable to other native amino acids than the proteinogenic amino acids selected for the development of this method. As peak shapes and signal-to-noise ratios could still be improved, further developments are wanted to upgrade this method. Due to the wide gradient (10 to 100% co-solvent in carbon dioxide), the method cannot truly be called either supercritical fluid chromatography (SFC) or enhanced-fluidity liquid chromatography (EFLC), but should be related to "unified chromatography" (UC), joining SFC and HPLC. Graphical abstract.

First inter-laboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities.

Supercritical Fluid Chromatography (SFC) has known a strong regain of interest for the last 10 years, especially in the field of pharmaceutical analysis. Besides the development and validation of the SFC method in one individual laboratory, it is also important to demonstrate its applicability and transferability to various laboratories around the world. Therefore, an inter-laboratory study was conducted and published for the first time in SFC, to assess method reproducibility, and evaluate whether this chromatographic technique could become a reference method for quality control (QC) laboratories. This study involved 19 participating laboratories from 4 continents and 9 different countries. It included 5 academic groups, 3 demonstration laboratories at analytical instrument companies, 10 pharmaceutical companies and 1 food company. In the initial analysis of the study results, consistencies within- and between-laboratories were deeply examined. In the subsequent analysis, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. The results obtained were compared with the literature values for liquid chromatography (LC) in the context of impurities determination. Repeatability and reproducibility variances were found to be similar or better than those described for LC methods, and highlighted the adequacy of the SFC method for QC analyses. The results demonstrated the excellent and robust quantitative performance of SFC. Consequently, this complementary technique is recognized on equal merit to other chromatographic techniques.

UHPLC method for multiproduct pharmaceutical analysis by Quality-by-Design.

An innovative Analytical Quality-by-Design (AQbD) methodology was followed to develop a specific and robust UHPLC method for the simultaneous separation of 16 active pharmaceutical ingredients (APIs). In the context of pharmaceutical repositioning, these molecules have been selected as good candidates for buccal per mucous (BPM®) administration route. Given the structural and physico-chemical diversity of compounds, an innovative development strategy based on QbD was applied. The main advantage of QbD is to ensure the robustness of the method. During a first scouting phase, the C18 chromatographic column was selected. Throughout the study, acetonitrile and ethanol based-mobile phases were investigated and compared. Ethanol was chosen as an alternative to acetonitrile due to its green properties coming from its lower toxicity and sourcing from renewable sources. Screening designs were performed to identify critical process parameters (CPPs). In ethanol media, temperature turned out to be a critical factor on peak retention and separation. Response surface methodology was then carried out to optimize CPPs and define the experimental domain of the method where complete separation between all peaks was obtained. Because changes in the elution order of the compounds occurred when modifying the experimental conditions, time differences between peaks were chosen as critical quality attributes, and an original data treatment was developed. It consisted in a systematic modelling of the time intervals between all possible pairs of peaks over the whole 3D experimental domain. Finally, a desirability analysis based on the smallest predicted time interval between peaks enabled to find optimal conditions only with ethanol based-mobile phases. Optimal conditions using ethanol, a Xbridge BEH Shield RP18 column and a 500 mL starting isocratic step, were determined by maximizing the desirability value and corresponded to a gradient slope of 2.57 %/min, a pH of 4.85, and a temperature of 33.7°C. A baseline separation of the 16 APIs was achieved with resolutions superior to 2.4 and the robustness of the method was experimentally validated.