Hyphenation of packed column supercritical fluid chromatography with mass spectrometry: where are we and what are the remaining challenges?

Over the last decades, packed column supercritical fluid chromatography (pSFC) using carbon dioxide (CO2) as supercritical fluid has gained interest as a complementary separation technique to liquid chromatography (LC). Various commercial solutions for the hyphenation to atmospheric pressure ionization (API) including electrospray (ESI) and atmospheric pressure chemical ionization (APCI) have been described using split-flow or full-flow introduction approaches. This review discusses various aspects of the hyphenation using these two approaches. It also illustrates the difference between LC-MS and SFC-MS. The benefits and challenges of the decoupling of the separation mobile phase from the ionization conditions are also pointed out. The effect of CO2 on ESI performance and the adduct reduction are also discussed. Finally, limitation of current hardware and the limited use of smaller column internal diameters (i.d.) are discussed. Graphical abstract.

Supercritical fluid chromatography-mass spectrometry using data independent acquisition for the analysis of polar metabolites in human urine.

The application of supercritical fluid chromatography with mass spectrometric (MS) detection (SFC-MS) was compared towards generic reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the analysis of urine with regards of ionization performance and analyte identification. The different chromatographic conditions were characterized with a selected set of 51 metabolites from different classes reported in the Human Metabolome DataBase (HMDB) and previously detected in human urine and/or plasma. SFC using a diol column with a gradient of carbon dioxide (CO2) and methanol with 10 mM ammonium hydroxide as modifier was able to retain and separate 20 polar analytes co-eluting in the RPLC eluent front. In the conditions investigated and compared to HILIC where many metabolites were also co-eluting, SFC showed a different ratio between elution domain and analysis time. Similar peak width and symmetry were observed, while retention time variability was slightly lower compared to that of HILIC (0.15% versus 0.24% and 1.26% for RPLC and HILIC, respectively). In SFC-MS, a significant signal enhancement (2-150 times, average of about 10 times) was measured after post-column make-up addition (MeOH/H2O, 95/5, v/v + 25 mM ammonium acetate) for 28 analytes. Nine analytes measured by LC-MS could not be detected in SFC-MS. Applicability of SFC-MS for metabolomics was investigated with the analysis of urine samples using data independent acquisition (DIA) and more specifically Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH/MS). Using a metabolomics library, 74 metabolites from human urine could be identified in positive mode in a single SFC-MS analysis of 15 min.

Effects of liquid post-column addition in electrospray ionization performance in supercritical fluid chromatography-mass spectrometry.

In supercritical fluid chromatography coupled to atmospheric pressure ionization mass spectrometry (SFC-MS), the use of a make-up post-column is almost mandatory to avoid analyte precipitation, especially when using low percentage of modifier (<5%) in the mobile phase. Due to the specific nature of gaseous CO2, the tuning of the make-up conditions in electrospray becomes an important factor and can be used to tune analyte sensitivity. Neither a dilution effect (loss of signal) nor a relevant degradation of chromatographic performances is observed with the addition of a make-up at various flow-rates, up to 0.7mL/min. From supercritical conditions (1mL/min 40°C, 150bar) to gaseous state (room temperature, atmospheric pressure), the CO2 expands around 430 times, contributing to almost 5% of the nebulizing process. In positive mode, the presence of ammonium ions either in the mobile phase or in the make-up did significantly increase the MS signal, even at basic apparent pH. The ionization performance of electrospray is influenced by the acidic buffer power of the carbon dioxide, and was found to be restricted in the apparent pH range of 3.8-7.2 in the various conditions investigated. This may challenge sensitive detection in negative mode, as illustrated for bosentan. The use of DMSO as make-up additive (up to 30%) showed a simplification of the full scan spectrum regarding the adducts. Finally, the optimization of make-up composition leads to an enhancement up to a factor of 69 on the electrospray MS response signal, for the SFC-SRM/MS analysis of HIV protease inhibitors in plasma extracted from Dried Plasma Spots.