Supercritical fluid chromatography coupled with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass spectrometry for compound speciation

•In-source hydrogen/deuterium exchange MS and SFC have been combined for the first time.•Combination is effective for speciation of N and O functional groups in mixture.•Combination is practical because only small of CH3OD was needed for the analysis.

An experimental setup for the speciation of compounds by hydrogen/deuterium exchange (HDX) with atmospheric pressure ionization while performing chromatographic separation is presented. The proposed experimental setup combines the high performance supercritical fluid chromatography (SFC) system that can be readily used as an inlet for mass spectrometry (MS) and atmospheric pressure photo ionization (APPI) or atmospheric pressure chemical ionization (APCI) HDX. This combination overcomes the limitation of an approach using conventional liquid chromatography (LC) by minimizing the amount of deuterium solvents used for separation. In the SFC separation, supercritical CO2 was used as a major component of the mobile phase, and methanol was used as a minor co-solvent. By using deuterated methanol (CH3OD), AP HDX was achieved during SFC separation. To prove the concept, thirty one nitrogen- and/or oxygen-containing standard compounds were analyzed by SFC-AP HDX MS. The compounds were successfully speciated from the obtained SFC-MS spectra. The exchange ions were observed with as low as 1% of CH3OD in the mobile phase, and separation could be performed within approximately 20 min using approximately 0.24 mL of CH3OD. The results showed that SFC separation and APPI/APCI HDX could be successfully performed using the suggested method.