Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC-MS-MS

摘要

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI-LC-MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroaceric acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C-18 and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column "TFA fix" using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1-36) in rat K(3)EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1-34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.