Chromatographie en phase supercritique : nouvelles perspectives dans le domaine de l'analyse pharmaceutique

L'attention prêtée aux préceptes de la chimie verte est en nette augmentation. Elle se traduit par de nombreux efforts consentis dans toutes les disciplines de la chimie afin minimiser leur impact environnemental. La chimie analytique n'y fait pas exception. Dans ce domaine, la contrainte relative à la hausse des coûts d'approvisionnement et de recyclage des solvants organiques, utilisés par la technique chromatographique de référence en phase liquide (LC), plaide également en faveur du recours à des options plus écologiques. La chromatographie en phase supercritique (SFC) constitue une alternative élégante puisqu'elle remplace la majeur partie des solvants par du CO2 pressurisé. La présente contribution se concentre sur l'évaluation des performances analytiques de la SFC portée par une instrumentation ultra-moderne. L'applicabilité de la SFC pour l'analyse d'un vaste spectre de composés pharmaceutiques ainsi que les modalités de couplage à la spectrométrie de masse (MS) y sont également explorées. Elles mettent en évidence à la fois la complémentarité, l'orthogonalité et la compétitivité de la SFC face à la LC, mais également son potentiel pour l'analyse de matrices biologiques

Evolution and Current Trends in Liquid and Supercritical Fluid Chromatography

The current trend in high performance liquid chromatography (HPLC) tends toward the achievement of higher separation efficiency and shorter analysis time. Indeed, better performance in LC has become increasingly important in recent years mainly driven by the challenges of either analyzing more complex samples or increasing the numbers of samples per time unit. In the recent development of particle technology, the use of fully porous sub-2 m particles and sub-3 m shell particles have received considerable attention. Beside packed columns, the new generation of silica-based monolithic columns also offers very high separation power. However, to take full advantage of these innovative phases, the chromatographic system has also to be drastically optimized in terms of upper pressure limit and system volume. This revolution in column technology now spreads and covers several modes of liquid chromatography such as reversedphase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), or even supercritical fluid chromatography (SFC). The HILIC and SFC, which can be considered as alternative modes of chromatography, could also be useful to extend the applicability of chromatography towards the analysis of very hydrophilic and lipophilic compounds, respectively. The present review gives an insight about the theory behind the success of current column technology and presents a summary of latest applications, using various modes of one-dimensional chromatography (RPLC, HILIC, SFC). This paper also shows that theoretically expected column efficiency could sometimes be compromised in practical work especially in the case of narrow bore columns

Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds

Currently, columns packed with sub-2 μm particles are widely employed in liquid chromatography but are scarcely used in supercritical fluid chromatography. The goal of the present study was to compare the performance, possibilities and limitations of both ultra-high performance liquid chromatography (UHPLC) and ultra-high performance supercritical fluid chromatography (UHPSFC) using columns packed with sub-2 μm particles. For this purpose, a kinetic evaluation was first performed, and van Deemter curves and pressure plots were constructed and compared for columns packed with hybrid silica stationary phases composed of 1.7 and 3.5 μm particles. As expected, the kinetic performance of the UHPSFC method was significantly better than that of the UHPLC. Indeed, the hmin values were in the same range with both strategies and were between 2.2 and 2.8, but uopt was increased by a factor of >4 in UHPSFC conditions. Another obvious advantage of UHPSFC over UHPLC is related to the generated backpressure, which is significantly lower in the presence of a supercritical or subcritical fluid. However, the upper pressure limit of the UHPSFC system was only ∼400 bar vs. ∼1000 bar in the UHPLC system, which prevents the use of highly organic mobile phases at high flow rates in UHPSFC. Second, the impact of reducing the particle size (from 3.5 to 1.7 μm) was evaluated in both UHPLC and UHPSFC conditions. The effect of frictional heating on the selectivity was demonstrated in UHPLC and that of fluid density or decompression cooling was highlighted in UHPSFC. However, in both cases, a change in selectivity was observed for only a limited number of compounds. Third, various types of column chemistries packed with 1.7 μm particles were evaluated in both UHPLC and UHPSFC conditions using a model mixture of acidic, neutral and basic compounds. It has been shown that more drastic changes in selectivity were obtained using UHPSFC columns compared to those obtained by changing UHPLC columns. In addition, there was a good complementarity between the two separation modes. Finally, by combining the use of small particles with supercritical fluids as a mobile phase, it was possible to achieve the analysis of pharmaceutical compounds in less than 1 min or to attain a peak capacity of more than 250 in approximately 40 min, both with a high degree of repeatability

The use of columns packed with sub-2 µm particles in supercritical fluid chromatography

The development of greener analytical techniques is a topic of great interest and in this context, supercritical fluid chromatography (SFC) is making an interesting comeback. The present review focuses on the latest developments of this technique. Improvements brought to the chromatographic systems and requirements to achieve full compatibility with columns packed with sub-2 µm particles (UHPSFC) are discussed. A thorough kinetic evaluation made using van Deemter representations, isopycnic and kinetic plots shows that performance achieved with state-of-the-art UHPSFC hardware and columns is comparable to that obtained in UHPLC. Orthogonal selectivity compared to reversed phase LC (RPLC) and extended selectivity modulation possibilities achievable in supercritical conditions using different stationary phase chemistries are presented. Finally, current applications of UHPSFC and its different hyphenation possibilities including mass spectrometry (MS) are also reviewed, in the hope of helping chromatography users to have a new look on the possibilities offered by this technique

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.)

Analysis of basic compounds by supercritical fluid chromatography: Attempts to improve peak shape and maintain mass spectrometry compatibility

While neutral and acidic compounds are well separated by upercritical fluid chromatography (SFC), basic analytes are more challenging to separate and often problems occur with their peak shapes. Two different methods were explored in the present paper to reduce these problems and maintain compatibility with mass spectrometry (MS). Five different, commercially available 2-ethylpyridine (2-EP) stationary phases were tested without a mobile phase additive using 92 pharmaceutical compounds with basic properties. The kinetic performances of the 5 columns were nearly identical, but the peak shapes of the basic drugs were strongly affected by the stationary phase. The PrincetonSFC 2-EP and Zymor Pegasus 2-EP phases clearly outperformed the other stationary phases, with 77% and 69% of the compounds having Gaussian peaks (and asymmetries between 0.8 and 1.4), respectively. Comparatively, the Waters Viridis Silica 2- EP, Waters Viridis BEH 2-EP and ES industries GreenSep 2-EP phases provided only 52%, 44% and 22% of the compounds with Gaussian peaks, respectively. These differences were attributed to the significant dissimilarities in their silica matrix properties. An alternative strategy was also performed with a hybrid silica stationary phase, Viridis BEH, using 20 mM ammonium hydroxide in the mobile phase, which was a mixture of CO2 and MeOH. With these conditions, 81% of the peaks observed for the basic analytes were Gaussian; however, this value dropped to 17% and 10% in the absence of additive and in the presence of 20 mM formic acid, respectively. Finally, the use of a hybrid bare silica stationary phase in the presence of 20 mM ammonium hydroxide is quite an interesting solution as this system is compatible with both ultra high performance SFC (UHPSFC) columns packed with sub-2 m particles and with MS detection. The overall applicability of this system was demonstrated with various mixtures of basic drugs

Interlaboratory study of a Supercritical Fluid Chromatography method for the determination of pharmaceutical impurities: evaluation of multi-systems reproducibility

Modern Supercritical Fluid Chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation, the present study extends the purpose to multi-instrumentations evaluation. In this context, three instrument types, namely Agilent®, Shimadzu®, Waters®, were included through 21 laboratories (n = 7 for each instrument). Firstly, method transfer was performed to assess the separation quality using these instruments and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Secondly, the inter-laboratory study was performed following the protocol defined by the sending lab. Analytical results were deeply examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Obviously, reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an ‘instrument type’ effect. Moreover, general speaking, the reproducibility variance was larger when considering all instruments than each type separately. The configuration of each modern SFC instrument easily explained the variability induced by the instrument. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods (i.e. reproducibility RSD around 15%). These results highlighted the robustness and the interest of SFC to provide accurate results for pharmaceutical quality control analysis

Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: From hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds

The recent market release of a new generation of supercritical fluid chromatography (SFC) instruments compatible with state-of-the-art columns packed with sub-2 m particles (UHPSFC) has contributed to the reemergence of interest in this technology at the analytical scale. However, to ensure performance competitiveness of this technique with modern analytical standards, a robust hyphenation of UHPSFC to mass spectrometry (MS) is mandatory. UHPSFC–MS hyphenation interface should be able to manage the compressibility of the SFC mobile phase and to preserve as much as possible the chromatographic separation integrity. Although several interfaces can be envisioned, each will have noticeable effects on chromatographic fidelity, flexibility and user-friendliness. In the present study, various interface configurations were evaluated in terms of their impact on chromatographic efficiency and MS detection sensitivity. An interface including a splitter and a make-up solvent inlet was found to be the best compromise and exhibited good detection sensitivity while maintaining more than 75% of the chromatographic efficiency. This interface was also the most versatile in terms of applicable analytical conditions. In addition, an accurate model of the fluidics behavior of this interface was created for a better understanding of the influence of chromatographic settings on its mode of operation. In the second part, the most influential experimental factors affecting MS detection sensitivity were identified and optimized using a design-of-experiment approach. The application of low capillary voltage and high desolvation temperature and drying gas flow rate were required for optimal ESI ionization and nebulization processes. The detection sensitivity achieved using the maximized UHPSFC–ESI-MS/MS conditions for a mixture of basic pharmaceutical compounds showed 4- to 10-fold improvements in peak intensity compared to the best performance achieved by UHPLC–ESI-MS/MS with the same MS detector