Abstract
Comprehensive two-dimensional liquid chromatography (LC × LC) is a powerful tool for the separation of complex biological samples. This technique offers the advantage of simplified automation and greater reproducibility in a shorter analysis time than off-line two-dimensional separation systems. In the present study, an LC × LC system is developed enabling simultaneous UV and MS detection, and which can be easily converted to a conventional reversed-phase LC-UV/MS system. In LC × LC, a 60-min reversed-phase LC separation with a linear solvent gradient in the first dimension is coupled to a second-dimension separation on a mixed-mode cation-exchange/reversed-phase column with a modulation time of 60 s. The isocratic separation in the second-dimension column is optimized by the use of a multi-step gradient where the organic and the ionic modifier are varied independently. Intraday ( n = 3) and interday ( n = 4) variability of the retention times were evaluated with the complete system and found to be 0.5% and 0.7%, respectively. Good linearity was observed in calibration curves for three different compounds varying in polarity.
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