Editorial

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SFC–MS versus RPLC–MS for drug analysis in biological samples “SFC–MS appears as a viable alternative to LC–MS for bioanalytical applications.” Keywords: MS • plasma • SFC–MS • supercritical fluid chromatography • urine

Brief overview of modern supercritical fluid chromatography & coupling of SFC with MS The advantages of supercritical fluid chromatography (SFC) over LC have been widely described in the past and include enhanced kinetic performance, due to the lower fluid viscosity and better solute diffusion coefficient; lower consumption of organic solvents; highly efficient chiral separation in presence of a supercritical fluid; and improvement of productivity at the preparative scale [1–3] . However, all these benefits were restricted by the lack of robustness/reliability, limited sensitivity and poor quantitative performance of old-generation SFC systems. This is certainly why SFC has never been considered as a forefront separation technique since its discovery in 1962 [4] . Over the last few years, a new generation of instruments and columns has appeared on the market. These new systems tackle the above-mentioned shortcomings. In addition, they also make SFC compatible with the most recent column types, such as the ones packed with fully porous sub-2 μm particles, or with sub-3 μm superficially porous particles [5] . When dealing with the analysis of biological samples, another important aspect to consider is compatibility of the separation technique with MS, as this detector allows improving selectivity, sensitivity and identification power [6] . In theory, the coupling of SFC with ESI source should be straightforward since the mobile phase is composed of a high proportion of CO2, which should enhance the evaporation step during the ionization process. However, it is also important

10.4155/BIO.15.41 © 2015 Future Science Ltd

to keep in mind that CO2 is decompressing through the transfer line between SFC and MS, leading to possible analyte precipitation and poor retention time repeatability. For these reasons, the coupling of SFC with ESI/MS involves the use of a dedicated interface. Among the existing interfaces, some of them are more universal or user friendly, while others are more sensitive [7] . Today, the most advantageous interface seems to be the pre-back pressure regulator (BPR) splitting interface using a make-up pump, as it offers good flexibility in terms of applicable chromatographic conditions, extended robustness and ease of use [7] . Which analytes are compatible with SFC–MS? In the early days of SFC, the mobile phase was exclusively composed of supercritical CO2, possessing a polarity close to that of hexane. Therefore, SFC was only suitable for the analysis of highly lipophilic compounds such as petroleum fractions, and remained hardly compatible with drugs and other compounds of biological interest [1,2] . To increase the mobile phase polarity and extend the range of compounds compatible with SFC, an organic modifier (in average between 5 and 40% methanol) is generally added to the supercritical CO2. With such a mobile phase, and considering the wide range of stationary phase chemistries available for SFC operation (e.g., polar, nonpolar, aromatic and mixedmode), this separation technique covers the application domain of different chromatographic modes including normal-phase LC, reversed phase LC (RPLC) and hydrophilic

Bioanalysis (2015) 7(10), 1193–1195

Vincent Desfontaine School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland

Lucie Nováková Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic

Davy Guillarme Author for correspondence: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland Tel.: +41 22 379 3463 Fax: +41 22 379 6808 davy.guillarme@ unige.ch

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Editorial  Desfontaine, Nováková & Guillarme interaction chromatography. As described in [6] , the range of analytes compatible with SFC is very broad since compounds possessing log P between -2 and >10 were successfully analyzed. In addition, several volatile mobile phase additives such as 10 mM ammonia, 10 mM ammonium formate and ammonium acetate, or even 2% water can be added to the mobile phase to promote peaks elution and symmetry of ionizable compounds, without compromising the MS detection [8,9] . In comparison to RPLC, which is the gold standard chromatographic method for bioanalytical applications, SFC offers an improved retention of hydrophilic compounds when selecting a polar stationary phase (e.g., bare silica, diol and amide) [10] , the possibility to elute very lipophilic substances (e.g., triglycerides and liposoluble vitamins) and appears as an orthogonal separation technique for the analysis of drugs and metabolites, due to the different retention mechanisms (retention mostly occurs through hydrophobic interactions and hydrogen bonding in RPLC and SFC, respectively) [1,2] . Which matrices and sample preparation techniques are suitable for SFC–MS? Up to now, SFC has been scarcely used for the analysis of complex biological matrices. Some years ago, it was mentioned that SFC was still in its adolescence for clinical applications, but had the potential to become a reference technique in a close future [11,12] . Over the years, the number of applications dealing with urine, plasma/blood or bile samples is growing but is still far from the amount of RPLC applications dealing with biological fluids [13,14] . Sample preparation step is usually mandatory prior to SFC–MS analysis, to achieve sufficient selectivity and sensitivity. Protein precipitation, SPE and LLE are obviously the most widely used. The use of any of these approaches prior to SFC–MS is quite straightforward, thanks to the use of organic solvent in the last step of the sample treatment. This includes the use of methanol or acetonitrile as an elution agent in SPE or the use of nonpolar solvents (i.e., heptane or ethyl acetate) in LLE. Due to the good compatibility of organic solvents as sample diluents in SFC–MS, the evaporation step, generally performed prior to RPLC–MS analysis, can be omitted, which saves both time and labor. One criticism when analyzing biological material in SFC–MS is related to the fact that some endogenous compounds (e.g., polar metabolites and residual proteins or enzymes) may not be eluted from the column, due to their strong interactions through H-bonding with the polar stationary phase commonly employed in SFC [15] . This is why it is essential to perform a

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suitable sample preparation able to eliminate as much undesired endogenous compounds as possible. Sensitivity achieved in SFC–MS versus LC–MS When dealing with the determination of drugs or metabolites in biological fluids, it is important to achieve sufficiently low LODs or LOQs. Therefore, it is important to verify that SFC–MS provides adequate sensitivity. In two recent studies [7,16] , it was demonstrated that the ESI source settings were relatively similar in SFC–MS and LC–MS. As shown in [16] , the presence of volatile salts (i.e., 10 mM ammonium formate) in the mobile phase does not compromise sensitivity in SFC–MS, while it could improve the peak shapes of ionizable compounds. Regarding the make-up solvent for successful SFC–MS operation, pure methanol or ethanol provides the highest sensitivity. In a recent paper, the LODs were compared in LC–MS and SFC–MS for 110 doping agents (acidic, basic and neutral) in urine matrix, after a simple dilute and shoot pretreatment [16] . With old-generation triple quadrupole MS/MS device, the achieved sensitivity was similar with both chromatographic techniques for about 27% of the doping agents, while it was enhanced in SFC–MS for 65% of the compounds. The better sensitivity achieved in SFC–MS versus LC–MS was attributed to a better desolvation of the mobile phase and to the absence of water in SFC–MS. On the other hand, the gain in sensitivity was more limited with a more recent MS/MS device of the same brand, (46% of substances offering similar sensitivity in LC–MS and SFC–MS and 38% of the doping agents with improved LODs in SFC vs LC). This behavior may be attributed to a better compatibility of modern MS/MS with highly aqueous RPLC mobile phases [16] . In conclusion, it is hard to assess whether the sensitivity could be improved in SFC–MS versus LC–MS, only on the basis of the compounds structure, as there are a lot of additional factors that also contribute. Nevertheless, the sensitivity is very often enhanced under SFC–MS conditions. Matrix effects in SFC–MS versus LC–MS Apart from sensitivity, another important parameter to consider in bioanalysis is the significance of matrix effects as it can result in poor accuracy, linearity, precision and sensitivity of the method. To date, the number of studies investigating matrix effects in SFC–MS is too limited and only one paper shows a systematic comparison of matrix effects in SFC–MS versus RPLC–MS for a wide range of compounds in urine matrix [16] . In this study, the occurrence of matrix effects was lower in SFC–MS versus LC–MS and no concentration dependence was observed. On

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SFC–MS versus RPLC–MS for drug analysis in biological samples

average, about 50% of the compounds were not prone to matrix effects in LC–MS, while this number grows to 70% in SFC–MS. In the case of urine, the salts and polar endogenous compounds responsible for serious matrix effects were eluted early in the LC–MS chromatogram, while they were more retained in SFC–MS, due to the very different retention mechanisms. This explains why the amount of matrix effects was different between both chromatographic techniques. There is a need to conduct similar experiments with other biological fluids, before we can draw reliable conclusions on the applicability of SFC–MS for bioanalysis. Conclusion In conclusion, SFC–MS appears as a viable alternative to LC–MS for bioanalytical applications. Indeed, it can be considered as a faster, greener, more universal and orthogonal analytical method, compared with References 1

Lesellier E, West C. The many faces of packed column supercritical fluid chromatography – A critical review. J. Chromatogr. A 1382, 2–46 (2015).

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Novakova L, Grand-Guillaume Perrenoud A, Francois I et al. Modern analytical SFC using columns packed with sub-2 μm particles: a tutorial. Anal. Chim. Acta 824, 18–35 (2014).

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Saito M. History of supercritical fluid chromatography: Instrumental development. J. Biosci. Bieng. 115(6), 590–599 (2013)

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Fekete S, Grand-Guillaume Perrenoud A, Guillarme D. Evolution and current trends in liquid chromatography. Curr. Chromatogr. 1, 15–40 (2014).

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Grand-Guillaume Perrenoud A, Veuthey JL, Guillarme D. The use of columns packed with sub-2 μm particles in supercritical fluid chromatography. Trends Anal. Chem. 63, 44–54 (2014).

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Kohler I, Guillarme D. Multi-target screening of biological samples using LC–MS/MS: focus on chromatographic innovations. Bioanalysis 6(9), 1255–1273 (2014)

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Grand-Guillaume Perrenoud A, Veuthey JL, Guillarme D. Coupling state-of-the-art supercritical fluid chromatography and mass spectrometry: from hyphenation interface optimization to high-sensitivity analysis of pharmaceutical compounds. J. Chromatogr A 1339, 174–184 (2014).

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Grand-Guillaume Perrenoud A, Veuthey JL, Guillarme D. Analysis of basic compounds by supercritical fluid chromatography: attempts to improve peak shape and maintain mass spectrometry compatibility. J. Chromatogr. A 1262, 205–213 (2012).

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Editorial

LC–MS. However, the number of studies showing a systematic comparison of sensitivity, matrix effects, linearity, precision and accuracy with different biological fluids in SFC–MS and LC–MS is still too limited to draw reliable conclusions. The preliminary data obtained on urine and discussed in this editorial prove that SFC–MS is often superior to LC–MS in these aspects. In the future, there is a need to extend this work to prove its applicability and to transform SFC–MS into a mature technique for bioanalysis. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.

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Novakova L, Grand-Guillaume Perrenoud A, Nicoli R et al. UHPSFC–MS/MS for screening of doping agents. I: Investigation of mobile phase and MS conditions. Anal. Chim. Acta 853, 637–646 (2015).

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Periat A, Grand-Guillaume Perrenoud A, Guillarme D. Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and their MS compatibility. J. Sep. Sci. 36, 3141–3151 (2013).

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Abbott E, Veenstra TD, Issaq HJ. Clinical and pharmaceutical applications of packed-column supercritical fluid chromatography. J. Sep. Sci. 31(8), 1223–1230 (2008)

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Rios A, Zougagh M, De Andres F. Bioanalytical applications using supercritical fluid techniques. Bioanalysis 2(1), 9–25 (2010).

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Lesellier E. Supercritical fluid chromatography for bioanalysis: practical and theoretical considerations. Bioanalysis 3(2), 125–131 (2011)

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Matsubara A, Fukusaki E, Bamba T. Metabolite analysis by supercritical fluid chromatography. Bioanalysis 2(1), 27–34 (2010)

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Spaggiari D, Mehl F, Desfontaine V et al. Comparison of liquid chromatography and supercritical fluid chromatography coupled to benchtop single quadrupole mass spectrometer for in vitro CYP-mediated metabolism assay. J. Chromatogr. A 1371, 244–256 (2014).

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Novakova L, Rentsch M, Grand-Guillaume Perrenoud A et al. UHPSFC–MS/MS for screening of doping agents. II: analysis of biological samples. Anal. Chim. Acta 853, 647–659 (2015).

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SFC-MS versus RPLC-MS for drug analysis in biological samples.

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