Splitless hyphenation of SFC with MS by APCI, APPI, and ESI exemplified by steroids as model compounds

A systematic evaluation of splitless hyphenation of supercritical fluid chromatography (SFC) with mass spectrometry (MS) was performed using different techniques for ambient pressure ionization. Interfaces commonly known from HPLC-MS/MS, i.e. electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI), were tested for their suitability in SFC-MS/MS. A triple quadrupole MS was used for data evaluation in a targeted multi-analyte design using endogenous steroids as model compounds. Individual optimization of the ionization parameters was performed in multi-dimensional design for best support of ionization in all three techniques. A post-column make-up was used to avoid analyte precipitation in the transfer capillary but also to support ionization independently from mobile phase composition. Buffer choice and concentration as well as temperature were found crucial in ESI and APCI. Best results for the multi-analyte method were obtained in both techniques using ammonium fluoride as make-up buffer. Instead of buffer solutions different organic solvents were used as dopants in APPI to support ionization. The mobile phase constituent isopropanol was already found to support ionization in APPI, however, for many analytes the addition of toluene resulted in superior results in terms of intensity. Comparing the optimized methods in terms of limit of detection (LOD), limit of quantification (LOQ), and sensitivity (slope of calibration curve) ESI was the best choice for the multiple analyte design. Only a few analytes resulted in a different optimum ionization, if focused on separately. In terms of linear dynamic range, APCI and APPI proved superior to ESI, where calibration over the whole range of concentrations (from LOD up to 5000 pg ∗ μL^-1) required quadratic regression.