期刊
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
卷 877, 期 20-21, 页码 1873-1881出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2009.05.012
关键词
Two-dimensional; HILIC; Peptide; LC-MS/MS; On-line extraction; Bioanalytical
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. (C) 2009 Elsevier B.V. All rights reserved.
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