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

• Published in: Journal of Chromatography A Vol. 1188; no. 2; pp. 216 - 226
• Main Authors: Eggink, Mark, Romero, Wilmar, Vreuls, René J., Lingeman, Henk, Niessen, Wilfried M.A., Irth, Hubertus
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.04.2008; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analysis; Analytical chemistry; Biological and medical sciences; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, Liquid - methods; Complex mixtures; Comprehensive two-dimensional liquid chromatography; Exact sciences and technology; General pharmacology; Hydrogen-Ion Concentration; LC × LC; Mass Spectrometry - methods; Medical sciences; Multi-step gradient elution; Organic Chemicals - isolation & purification; Other chromatographic methods; Pharmacology. Drug treatments; Time Factors; Ultraviolet Rays; LC × LC; Multi-step gradient elution; Complex mixtures; Comprehensive two-dimensional liquid chromatography; Human; Drug; Urine; LC x LC; Biological fluid; Elution; Gradient; Chemical analysis; Two dimensional chromatography; Atmospheric pressure; Multicomponent mixture; Coupled method; Elimination; HPLC chromatography; Chemical ionization; Optimization; Reversed phase chromatography; Ultraviolet detector; Mass spectrometry
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2008.02.072

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.