Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography–mass spectrometry setup

A generic LC–MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1–34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1 vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC–MS setup, large volumes of filtrates (e.g., 2 × 750 μL) were injected and the peptides were trapped on a 1 mm i.d. × 10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3 mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

► A generic LC–MS protocol to develop methods for quantification of peptides.
► An original 2-step sample clean-up: protein precipitation and ultrafiltration.
► Nine human peptides tested successfully with excellent extraction recoveries.
► A specific column switching LC setup with 2 refocalization of peptides on columns.
► Limits of detection in the mid-picomolar range with an 8-year-old MS instrument.