Utilisation de la chromatographie d'interaction hydrophile pour l'analyse de composés d'intérêt pharmaceutique

L'intérêt pour les composés hydrophiles est devenu particulièrement important ces dernières années, suite à l'importance de la métabolomique et de l'analyse des modifications post-traductionnelles des protéines, comme la glycosylation. De plus, de nombreuses substances d'intérêts pharmaceutiques sont très hydrophiles, comme les acides aminés, les neurotransmetteurs, les oligosaccharides, les nucléosides, ainsi que certains principes actifs et leurs métabolites. L'analyse de ce type de composés reste complexe, car la chromatographie liquide à polarité des phases inversées (RPLC) n'est pas adaptée à de tels analytes. La chromatographie d'interaction hydrophile (HILIC) est une technique de choix permettant l'analyse de composés hydrophiles non retenus en RPLC. Le mode HILIC permet également la rétention de composés ionisables et offre une excellente compatibilité avec la spectrométrie de masse (MS). Ainsi, cette technique est appropriée pour l'analyse d'un grand nombre de composés d'intérêt pharmaceutique. Les différentes possibilités et limites de ce mode chromatographique ont été évaluées

Screening of the most relevant parameters for method development in ultra-high performance hydrophilic interaction chromatography

The goal of the present work was to provide some guidelines for method development in hydrophilic interaction chromatography (HILIC). For this purpose, a training set of 82 representative pharmaceutical compounds possessing diverse polarity and including acidic, basic and neutral properties was analyzed. All these drugs were injected on five short HILIC columns packed with sub-2 µm particles and dedicated for UHPLC (ultra-high performance liquid chromatography) operation. Four different pH conditions ranging from pH 3 to 6 were tested at two ionic strengths (10 and 50 mmol/L) and finally, the reference organic modifier in HILIC, namely acetonitrile was modified with small amounts of methanol or isopropanol. From these experiments and using multivariate data analysis, it is clear that the stationary phase was the most relevant parameters for tuning selectivity in HILIC, since the types of interactions (i.e. dipole–dipole, hydrogen bonding and ion exchange) with analytes strongly vary between columns. Among the selected phases, the diol phase was the less interesting one, in terms of selectivity and peak shape. The zwitterionic phase was attractive, as it allowed a better retention of acidic compounds. Finally, the bare silica phase was the most versatile HILIC column packed with sub-2 µm particles in terms of retention, peak shape and selectivity. Mobile phase pH was the other important parameter to achieve an appropriate selectivity and retention, even if it remains always difficult to assess precisely the mobile phase pH, analyte pKa and silanols pKa, when working with more 70% acetonitrile. Finally, buffer ionic strength and organic modifier nature could be considered as secondary parameters for HILIC method development. In conclusion, screening four different columns packed with sub-2 µm particles at two mobile phase pH, using a fast gradient seems to be a good generic approach for initial HILIC method development. The total time for such a screening was estimated at ∼1 h, including reequilibration

Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS compatibility

The goal of this study was to compare the performance of three separation techniques for the analysis of 57 hydrophilic compounds. RPLC, hydrophilic interaction liquid chromatography (HILIC) and subcritical fluid chromatography (SFC) were tested. The comparison was based on the retention, selectivity, peak shape (asymmetry and peak width) and MS sensitivity. As expected, RPLC had some obvious limitations for such classes of compounds, and on average the %ACN required to elute these hydrophilic substances was 4, 7, and 11% ACN at pH 3, 6, and 9, respectively. However, a hybrid polar-embedded C18 phase with an appropriate mobile phase could represent a viable strategy for hydrophilic basic compounds with log D greater than -2 on average. HILIC and SFC were found to be more appropriate for analyzing a large majority of these hydrophilic analytes (~60 and 70% of compounds eluted during the gradient in HILIC and SFC), while maintaining good MS sensitivity. Finally, this work demonstrated the complementarity of the three analytical techniques and showed that the selection of a suitable strategy should mostly be based on physicochemical properties of the analytes (pKa, log D, H-bonding capability, etc.)

Advances in Hydrophilic Interaction Liquid Chromatography for Pharmaceutical Analysis

Hydrophilic interaction liquid chromatography (HILIC) has recently become more important, particularly for the analysis of polar drugs, metabolites and biologically relevant compounds in glycomics, proteomics, metabolomics and clinical analysis. HILIC makes it possible to increase the retention of polar coumpounds, achieve orthogonal selectivity and increase mass spectrometry (MS) sensitivity, compared with reversed-phase liquid chromatography. This article discusses the advantges and limitations of HILIC in a variety of practical applications in the pharmaceutical industry

Potential of hydrophilic interaction chromatography for the analyticalcharacterization of protein biopharmaceuticals

A new stationary phase based on wide-pore hybrid silica bonded with amide ligand has been used toexplore the utility of HILIC for the analytical characterization of protein biopharmaceuticals. Various,highly-relevant samples were tested, including different insulins, interferon a-2b and trastuzumab. Thiswork shows that HILIC can be successfully employed for the analysis of therapeutic proteins and mAbs,using mobile phase compositions comprised of between 65 and 80% ACN and 0.1% TFA. In terms of elutionorder and selectivity, these HILIC separations have proven to be highly orthogonal to RPLC, while thekinetic performance remains comparable. In the case of characterizing trastuzumab, HILIC was uniquelyable to resolve several important glycoforms at the middle-up level of analysis (fragments of 25–100 kDa).Such a separation of glycoforms has been elusive by other separation mechanisms, such as RPLC and IEX.Besides showing orthogonality to RPLC and improved separations of glycoforms, HILIC offers severaladditional benefits for biopharmaceutical characterization: i) an inherent compatibility with MS, ii) areduced requirement for very high mobile phase temperatures that are otherwise needed in RPLC tolimit undesirably strong adsorption to the surface of the stationary phase, and iii) the possibility tocouple several columns in series to improve resolving power, thanks to comparatively low mobile phase viscosity

Optimized selection of liquid chromatography conditions for widerange analysis of natural compounds

Plant secondary metabolites are an almost unlimited reservoir of potential bioactive compounds. In viewof the wide chemical space covered by natural compounds, their comprehensive analysis requires mul-tiple and complementary approaches. In this study, numerous chromatographic conditions were testedfor the analysis of a set of 120 representative natural compounds covering a wide polarity range (18log P units). The experiments were performed on 59 different conditions involving 29 RPLC and HILICdedicated stationary phases, as well as more exotic mixed mode columns. The best RPLC and HILIC con-ditions were determined using Derringer’s desirability functions, based on various criteria (i.e. retention,peak shape, distribution of compounds during the gradient. . .). After this first selection, only the mostpromising conditions were kept (19 in RPLC and 11 in HILIC). The selectivity complementarity amongeach chromatographic mode was assessed by principal component analysis (PCA) and hierarchical clusteranalysis (HCA). In RPLC, a pentabromobenzyl (PBrBz) stationary phase was identified as particularly ver-satile and could constitute an elegant first intention screening column. Two additional conditions allowedto extend the range of natural compounds space that can be analyzed, while offering better selectivityfor basic analytes (hybrid silica graft with C8 moiety operated at pH 9 (Hyb C8)) and acidic compounds(positively charged hybrid silica graft with pentafluorophenyl moiety (Hyb+ PFPh). Although less genericin terms of amenable compounds, an ion exchange/RP mixed mode stationary phase (MM TriP1) offerednotably enhanced retention of more polar analytes under RPLC conditions. With these four conditions,89% of the natural substances were detected by LC–MS with acceptable retentions and peak shapes. InHILIC, four acceptable and complementary conditions were also highlighted. Both Syncro-Z (zwitterionicHILIC phase) and Diol columns were found to offer balanced retention and selectivity for most of thepolar compounds (log DpH3< 1.0). These two columns could be advantageously complemented by hybridAmide column operated at pH 3 and Amino stationary phase at pH 5, to further enhance both retentionand selectivity of polar basic and acidic species, respectively