Please cite this article in press as: H.R. Sobhi, et al., J. Chromatogr. A (2011), doi:10.1016/j.chroma.2011.09.072 ARTICLE IN PRESS G Model CHROMA-352575; No. of Pages 8 Journal of Chromatography A, xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography–mass spectrometry setup Hamid Reza Sobhi a,b,1 , Bilgin Vatansever a,c,d,1 , Arno Wortmann e , Eric Grouzmann c , Bertrand Rochat a,∗ a Quantitative Mass Spectrometry Facility, University Hospital of Lausanne, CHUV, BH18-228, 1011 Lausanne, Switzerland b Department of Chemistry, Tehran Payamenoor University, Tehran, Iran c Clinical Pharmacology, University Hospital of Lausanne, CHUV, Lausanne, Switzerland d Non-Clinical Development DMPK, Global Bioanalytics, Bioanalytical NCE Laboratory, Merck Serono, Colleretto Giacosa (TO), Italy e Analytical and Formulation Development, F. Hoffmann-La Roche Ltd., Basel, Switzerland a r t i c l e i n f o Article history: Received 1 September 2011 Received in revised form 23 September 2011 Accepted 26 September 2011 Available online xxx Keywords: LC–MS Peptide Absolute quantification Ultrafiltration Extraction protocol a b s t r a c t 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 -melanotropin (-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 L) 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The absolute quantification of native or therapeutic pep- tides is crucial when studying their fate and attempting to understand their pharmacological action(s). Indeed, the kinetics of peptides in plasma or tissue is controlled by the activi- ties of many proteases/peptidases [1,2]. Proteases will generate peptides from proteins or pro-peptides and will further trans- form them into active or inactive fragments underlying the proteome–protease–peptidome relationship and peptidomics con- cept [1–3]. Therefore, it is necessary to quantify peptides with accuracy and specificity. During the last few decades, immunoassays have been the stan- dard methodology for the sensitive quantification of peptides in biomatrices. Until recently mass spectrometry has always been ∗ Corresponding author. Tel.: +41 21 314 41 58; fax: +41 21 314 42 88. E-mail address: bertrand.rochat@chuv.ch (B. Rochat). URL: http://www.unil.ch/qmsf (B. Rochat). 1 Both authors have contributed equally to this work. considered to be less sensitive than immunoassays, especially for the quantification of large peptides (>2000 amu). However, recent developments in LC–MS technology have challenged this idea. In addition, the use of antibodies that are not fully able to distinguish between peptidic fragments derived from the same native peptide, can explain the limitations of many immunoassays when applied to pharmacokinetic studies of peptides. This lack of assay specificity generally results in poor pharmacokinetic–pharmacodynamic (PK–PD) relationships. Therefore, mass spectrometry has a key role to play in the dynamic study of the peptidome since it can achieve specific identification and absolute quantification (isotopic dilution assay) of peptides. The peptidome, composed of thousands of native peptides, is still a terra incognita [1] and is a very promising field of investigation in attempting to discover biomarkers and signaling messengers [1,2,4]. However, there is still a need for a generic analyti- cal approach for quantitative peptidomics including hydrophobic and hydrophilic peptides. At present, the development of sen- sitive methods for peptide determination can be dramatically time consuming. This becomes apparent when considering that the peptidome is mostly composed of large (15–50 amino acids; 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.09.072