Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC-MS-MS.
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 trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C18 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.
Liu
Ang
A
College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Tweed
Joseph
J
Wujcik
Chad E
CE
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009862:Online Systems; D044162:Parathyroid Hormone-Related Protein / Q000097:blood*; D010446:Peptide Fragments / Q000097:blood*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014269:Trifluoroacetic Acid / Q000737:chemistry
D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; C079957:parathyroid hormone-related peptide (1-36); D014269:Trifluoroacetic Acid