Development and optimization of a system for comprehensive two-dimensional liquid chromatography with UV and mass spectrometric detection for the separation of complex samples by multi-step gradient elution.
Comprehensive two-dimensional liquid chromatography (LC x 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 x 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 x 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 60s. 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.
Eggink
Mark
M
VU University Amsterdam, Faculty of Science, Department of Analytical Chemistry and Applied Spectroscopy, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. meggink@few.vu.nl
Romero
Wilmar
W
Vreuls
René J
RJ
Lingeman
Henk
H
Niessen
Wilfried M A
WM
Irth
Hubertus
H
D002853:Chromatography, Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D009930:Organic Chemicals / Q000302:isolation & purification*; D013997:Time Factors; D014466:Ultraviolet Rays
D009930:Organic Chemicals