Alternative Strategies to Reversed-Phase Liquid Chromatography for the Analysis of Pharmaceutical Compounds

• Shimadzu Scientific Instruments

Abstract

Reversed phase liquid chromatography (RPLC) is the gold standard analytical strategy in the pharmaceutical industry. In this contribution, we will describe two alternative chromatographic approaches, namely hydrophilic interaction chromatography (HILIC) and supercritical fluid chromatography (SFC), which are more and more commonly used in pharmaceutical analysis. Among their advantages, these two strategies offer better retention of polar substances, alternative selectivities and enhanced MS sensitivity, in comparison with RPLC.

Keywords

Supercritical fluid chromatography, hydrophilic interaction chroma-tography, SFC, HILIC

Introduction

Because the analysis of drugs is required at every stage of the drug development process, there is a need for powerful analytical methods. Reversed phase liquid chromatography (RPLC) is considered as the gold standard and is routinely applied in the pharmaceutical industry, due to its robustness, versatility and high resolving power.

However, as depicted in Figure 1, RPLC is only appropriate for a limited range of compounds, having log P (partitioning coefficient in octanol/water mixture) comprised between -1 and 7. When analyzing more hydrophilic substances (log P < -1), the retention is not sufficient under RPLC conditions using C18 stationary phase. For such very polar molecules, ion exchange chromatography (IEX) or ion pairing chromatography (IPC) can be applied, but these approaches suffer from poor kinetic performance (IEX), lack of robustness (IPC) and difficult hyphenation with mass spectrometry (IEX and IPC). As discussed in the next section, hydrophilic interaction chromatography (HILIC) and supercritical fluid chromatography (SFC) may represent better alternatives. On the other hand, the most lipophilic compounds (log P > 7) cannot be eluted from the C18 stationary phase using common mobile phase consisting in a mixture of organic solvents (acetonitrile or methanol) and buffered water. Normal phase liquid chromatography (NPLC) has been applied in the past, but it requires the use of toxic and expensive solvents and is not as robust as RPLC. Again, SFC may represent a suitable alternative, as illustrated in this review paper.³

Figure 1. Applicability of different chromatographic modes for a wide range of biological and pharmaceutical compounds possessing log P between -10 and 10.

Except the issue related to retention in RPLC, there is also often a need for orthogonal methods able to offer alternative selectivity in the pharmaceutical industry. As an example, if a suitable separation of active pharmaceutical ingredient (API) and related impurities cannot be achieved in RPLC, HILIC and SFC can be considered to modify the elution order and overall separation quality. In addition, having a second method at disposal, orthogonal to the first one, may be beneficial for confirmation purpose.

Finally, there is a need for highly sensitive analytical method involving mass spectrometry (MS) in bioanalysis. Then, it has been proved that replacing RPLC by HILIC or SFC could be a way to enhance sensitivity.

The beneficial features of HILIC and SFC will be exposed in details in the present review.

Hydrophilic Interaction Chromatography (HILIC)

HILIC is a chromatographic mode that is gaining in importance for the last few years, particularly with the growing need for analyzing a large variety of biologically active substances including pharmaceutical compounds, amino acids, peptides, neurotransmitters, oligosaccharides, carbohydrates, nucleotides, or nucleosides.¹ HILIC is characterized by a hydrophilic stationary phase (similar to NPLC) and an aqueous organic solvent as mobile phase, containing more than 70% of aprotic organic solvent (often acetonitrile), and can be applied to polar and/or ionizable compounds (similar to IEX).²

The main advantage of HILIC is its ability to retain hydrophilic substances without the need for toxic and expensive solvents such as those commonly employed in NPLC, and without important amount of salts and ion pairing reagents, hardly compatible with MS detection.³

Besides hydrophilic compounds, HILIC can also be considered as a complementary (orthogonal) technique to RPLC for the analysis of any type of ionizable compounds, including drugs. Indeed, the elution order is almost reversed in HILIC (hydrophilic partitioning) compared to RPLC (hydrophobic partitioning).⁴ In addition, due to the strong contribution of the ion exchange mechanism, the obtained selectivity is generally very different in HILIC compared to RPLC. This is, for example, illustrated in Figure 2, demonstrating the separation of nine model peptides under RPLC and HILIC conditions.⁵ As shown, the peaks 5 and 6 eluted closely under RPLC conditions, while these two substances were eluted as the first and last compounds under HILIC conditions. Obviously, the reverse situation could also be observed, and for example, peaks 1 and 3 were very well separated in RPLC, while their separation remained difficult in HILIC.

Figure 2. Separation of 9 model peptides having molecular weights between 1 and 9 kDa. The separations were performed on 150 x 2.1mm columns packed with 1.7 µm particles.

Another important benefit of HILIC is the enhanced signal when using MS detection.^6,7 This behavior can be explained by the more efficient desolvation of highly organic mobile phases (low surface tension and density of ACN compared to water). As illustrated in Figure 3, a significant improvement in sensitivity can be achieved. For a range of about 50 drugs, HILIC-ESI-MS/MS was more sensitive than RPLC-ESI-MS/MS for 89% of the compounds.⁶ In average, the gain in sensitivity was equal to 10, but in particular cases, it can be more than 30-fold, depending on the nature of the compounds and the mobile phase composition. It is also important to notice that this sensitivity improvement is strongly dependent on the ionization source geometry.⁷

Figure 3. MS sensitivity improvement between RPLC and HILIC conditions for about 50 pharmaceutical compounds using a mobile phase pH of 3.

Finally, due to the low viscosity of the highly organic mobile phase employed in HILIC (2-3 times lower than RPLC), the generated backpressure is also limited. This allows for working at a higher flow rate with longer columns and smaller particle sizes to further improve kinetic performance.⁸

Thanks to the above mentioned advantages, numerous pharmaceutical applications were reported in HILIC over the last few years, including the analysis of i) drug compounds, ii) their impurities and iii) metabolites in bulk material, pharmaceutical formulations or body fluids (blood, plasma, serum, and urine). There are a number of very good reviews which summarize these applications.^9,10

Supercritical Fluid Chromatography (SFC)

SFC is a separation technique first described in 1962 by Klesper, which consists in using a fluid (generally CO₂ ) in its supercritical state (ΔP and T beyond its critical point) as main mobile phase component.¹¹ Under supercritical conditions, the viscosity and diffusivity of such a fluid is very close to those of a gas, resulting in excellent separation efficiency at high mobile phase velocity, while maintaining a low pressure.¹² The exceptional kinetic performance of SFC is illustrated in Figure 4, comparing van Deemter curves and pressure plots for regular HPLC, UHPLC, regular SFC and UHPSFC (ultra-high performance SFC).¹³ Besides the very good kinetic performance, the density and solvating power, similar to the one of a liquid, also provide good solubility and fast analytes transportation.

Figure 4. Kinetic performance of SFC and UHPSFC vs. HPLC and UHPLC. (A) van Deemter curves, (B) Pressure plots.

Despite these interesting features, the pharmaceutical industry showed limited interest for SFC in its early time, and continued to use the well-established liquid and gas chromatography (LC and GC). In the last few years, there has been a renewed interest in SFC, stimulated by the introduction of a new generation of instruments and columns from the major providers of chromatographic material. The known previous limitations of SFC, such as weak UV sensitivity, limited reliability and poor quantitative performance have been tackled with these advanced instruments.^14,15

It is also important to notice that modern packed-column SFC instruments are very close to LC systems. Only a few differences can be highlighted: i) the pumping system should be cooled down to about 4°C, to pump CO2 under a liquid state, ii) the UV cell has to withstand a pressure of up to 400 bar, iii) at the detector outlet, a backpressure regulator has to be added to maintain a sufficient pressure within the system, iv) a polar stationary phase is commonly used for pharmaceutical applications, but apolar and/or aromatic phases can also be used depending on the physico-chemical properties of the compounds to be analyzed.

Due to the obvious benefits of SFC in terms of kinetic performance and its complementarity to LC, modern SFC represents today an additional strategy in the toolbox of the analytical scientist, which may be particularly interesting in pharmaceutical analysis.¹⁶Figure 5 shows the potential of SFC vs. RPLC for the analysis of seven steroids. Because the interaction mechanism is totally changed between RPLC (retention is mostly driven by hydrophobic interaction) and SFC (retention is typically controlled by H-bond capability of the analytes and stationary phases), the retention order and selectivities were very different between both modes, while the peak shapes and analysis times remain comparable. This example proves that SFC can be used to analyse the same type of substances as the ones analysed in RPLC (See Figure 1).

Figure 5. Separation of 7 steroids in UHPSFC and UHPLC using columns packed with 1.7µm particles.

In addition, Figure 6 shows the applicability of SFC for the analysis of much more hydrophilic and lipophilic substances (water and fatsoluble vitamins), which cannot be easily analyzed under regular HPLC conditions.¹⁷ To achieve this extreme SFC separation covering a wide range of log P values, the amount of MeOH (co-solvent) has to be changed during the experiment from 2 to 100 %MeOH. At the beginning of the gradient, the liposoluble vitamins were eluted in presence of a large proportion of supercritical CO2 (apolar solvent). On the other hand, the hydrosoluble vitamins were only eluted with a large proportion of MeOH (up to 100% MeOH).¹⁷ Due to the presence of a very large proportion of methanol, the technique can no longer be called SFC since the fluid is not under a supercritical state. However, such conditions can be successfully applied for the analysis of a wide range of substances with the same equipment and similar mobile phase components.

Figure 6. Extreme SFC conditions using a gradient from 2 to 100% MeOH in CO2 for the analysis of liposoluble and hydrosoluble vitamins within the same run.

It is finally worth mentioning that SFC can be combined with MS, but a specially designed interface has to be employed to limit the risk of analytes precipitation caused by the decompression of CO₂ and to attain sufficient ionization yield.¹⁸ Today, several commercial SFC-MS interfaces allow highly sensitive and robust SFC-MS operation. SFC-MS generally offers better sensitivity, and lower impact of MS operating conditions on sensitivity (better robustness) vs. HPLC-MS. This behavior is related to the absence of water in the mobile phase and the fact that CO₂ can be easily eliminated in the ionization source. SFC-MS(/MS) was successfully employed in various fields including lipidomics, metabolomics, bioanalysis, food and environmental analysis.¹⁹

Based on all the positive features of SFC described in this contribution, it is logical that numerous applications report the use of SFC for the analysis of chiral and achiral pharmaceutical products at the analytical and preparative scales. For more information on applications of SFC in the pharmaceutical field, the readers can refer to the following review papers.^20,21

Summary and Conclusions

The growing number of applications in HILIC highlights the rising interest for this technique. HILIC offers some obvious benefits compared to RPLC for the analysis of drugs in formulations or in biological fluids, including (i) higher retention of polar drugs and metabolites, (ii) orthogonal or complementary selectivity to RPLC for pharmaceutical compounds, and (iii) improved MS sensitivity in bioanalytical applications.

SFC also appears as another promising strategy in the pharmaceutical analysis. Compared to RPLC, which is still considered as the gold standard, SFC technology offers the following benefits: i) equivalent or better kinetic performance (throughput and/or plate count) than modern RPLC, ii) extension of the range of compounds compatible with SFC towards more hydrophilic and also highly lipophilic substances, without changing mobile phase constituents, iii) high orthogonality between SFC and RPLC, iv) SFC is also a greener approach compared to RPLC as the consumption of organic solvents and waste generation may be reduced, v) improved MS sensitivity in SFC-MS vs. RPLC-MS.

Despite these obvious advantages, HILIC and SFC are still not as well described as RPLC and some reluctance to adopt these techniques may be observed in the pharmaceutical industry. The situation has recently begun to change and in the future, we can expect more and more fully validated HILIC or SFC methods applied in routine analysis.

References

1. Buszewski B, Noga S. Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique. Anal. Bioanal. Chem. 2012;402(1): 231-247.
2. Periat A, Debrus B, Rudaz S, Guillarme D. Screening of the most relevant parameters for method development in ultra-high performance hydrophilic interaction chromatography. J. Chromatogr. A 2013;1282:72-83.
3. 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. 2013;36:3141-3151.
4. Hemström P, Irgum K. Hydrophilic interaction chromatography. J. Sep. Sci. 2006; 29(12): 1784-1821.
5. Ruta J, Rudaz S, McCalley DV, Veuthey JL, Guillarme D. A systematic investigation of the effect of sample diluent on peak shape in Hydrophilic Interaction Liquid Chromatography. J. Chromatogr. A 2010;1217(52):8230-8240.
6. Periat A, Boccard J, Veuthey JL, Rudaz S, Guillarme D. Systematic comparison of sensitivity between hydrophilic liquid chromatography and reversed phase liquid chromatography coupled with mass spectrometry. J. Chromatogr. A 2013;1312:49-57.
7. Periat A, Kohler I, Bugey A, Bieri S, Versace F, Staub C et al. Hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry: effect of electrospray ionization source geometry on sensitivity. J. Chromatogr. A 2014;1356:211-220
8. Heaton JC, McCalley DV. Comparison of the kinetic performance and retentivity of sub- 2 μm core–shell, hybrid and conventional bare silica phases in hydrophilic interaction chromatography. J. Chromatogr. A 2014;1371:106-116.
9. Dejaegher B, Vander Heyden Y. HILIC methods in pharmaceutical analysis. J. Sep. Sci. 2010;33(6-7):698-715.
10. Hsieh Y. Potential of HILIC-MS in quantitative bioanalysis of drugs and drug metabolites. J. Sep. Sci. 2008;31(9):1481-1491.
11. Lesellier E, West C. The many faces of packed column supercritical fluid chromatography--a critical review. J. Chromatogr. A 2015;1382:2-46.
12. Fekete S, Grand-Guillaume Perrenoud A, Guillarme D. Evolution and current trends in liquid chromatography. Curr. Chromatogr. 2014;1:15-40.
13. Grand-Guillaume Perrenoud A, Veuthey JL, Guillarme D. Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography. J. Chromatogr. A 2012;1266:158-167.
14. Novakova L, Grand-Guillaume Perrenoud A, Francois I, West C, Lesellier E, Guillarme D. Modern analytical SFC using columns packed with sub-2 µm particles: A tutorial. Anal. Chim. Acta 2014;824:18-35.
15. Dispas A, Desfontaine V, Andri B, Lebrun P, Kotoni D, Clarke A, et al. Quantitative determination of salbutamol sulfate impurities using achiral Supercritical Fluid Chromatography. J. Pharm. Biomed. Anal. 2017;134:170-180.
16. 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. 2014;63: 44-54.
17. Taguchi K, Fukusaki E, Bamba T. Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography. J. Chromatogr. A 2014;1362:270-277.
18. 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 2014;1339:174-184.
19. Desfontaine V, Veuthey JL, Guillarme D. Hyphenated detectors: mass spectrometry [In] Supercritical fluid chromatography. C.F. Poole, Editor, Elsevier, 2017, In press
20. Desfontaine V, Guillarme D, Francotte E, Novakova L. Supercritical fluid chromatography in pharmaceutical analysis. J. Pharm. Biomed. Anal. 2015;113:56-71.
21. Lemasson E, Bertin S, West C. The use and practice of achiral and chiral supercritical fluid chromatography in pharmaceutical analysis and purification. J. Sep. Sci. 2016;39(1):212-233.