Direct coupling of supercritical fluid chromatography with tandem mass spectrometry for the analysis of amino acids and related compounds: Comparing electrospray ionization and atmospheric pressure chemical ionization

- Direct coupling of supercritical fluid chromatography (SFC) with MS/MS.
- Comparison of the ionization performance using APCI or ESI.
- Separation of amino acids and other relevant metabolites.

For acceptance of supercritical fluid chromatography (SFC) as a routine analysis method, the hyphenation to mass spectrometry (MS), which is typically achieved either by a splitting device or by the employment of an additional make up flow, has to be improved. Direct coupling of SFC to MS (/MS) would simplify the handling of this method. Consequently, this work focused on the direct coupling of SFC to mass spectrometry and the influence of the employed ion source on signal intensities of polar and ionic compounds in biological samples. A method for separating metabolites of the tryptophan pathway as well as other amino acids is shown. Results demonstrate that SFC is capable of separating analytes of polar and ionic nature. Modifications of the SFC system by cryostat cooling lead to higher temperature stability in the booster pump and therefore to a better reproducibility of retention times and a low dispersion nozzle inside the active back pressure regulator (ABPR) significantly improves peak shape and sensitivity when using the MS. The comparison of the ionization efficiencies using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) in positive and negative ion mode shows analyte depending sensitivities. However, results indicate that APCI is better suitable for the ionization of amino acids with polar side chains, whereas ESI proved superior for the ionization of amino acids featuring hydrophobic residues. Analyte signals were suppressed with ESI when using a complex matrix such as human serum, but rather enhanced when using APCI.

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