The serial coupling of reversed phase and hydrophilic interaction liquid chromatography (RPLC/ HILIC) with mass spectrometric detection (MS) is emerging as a versatile and reliable technique for the comprehensive analysis of complex samples. The connection of a HILIC column in series to a RPLC one broadens the elution window, with the effect that polar and apolar solutes can be simultaneously separated. In order to convert already running RPLC systems into more comprehensive tools, it is necessary to know how the addition of a HILIC column influences the RPLC separation. For this purpose, the behavior of apolar analytes in serial RPLC/HILIC systems with MS detection was investigated. The RPLC separation was performed on C18 material, coupled with five HILIC phases, respectively, under different elution conditions regarding salt content and pH of the HILIC mobile phase. The retention factors of the selected analytes systematically increased with the following HILIC phases in the order phosphorylcholine < sulfobetaine < diol < silica phase. In most cases, the HILIC mobile phase conditions did not affect the selectivity of the apolar solutes (the RSDs of the retention factors were in general below 0.9%), but they influenced the intensity of the MS signals.
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