Simultaneous Absolute Quantification of the Glucose-Dependent Insulinotropic Polypeptides GIP 1-42 and GIP 3-42 in Mouse Plasma by LC/ESI-MS/MS: Preclinical Evaluation of DP-IV Inhibitors Alexandros P. Siskos, † Theodora Katsila, † Evangelos Balafas, ‡ Nikolaos Kostomitsopoulos, ‡ and Constantin Tamvakopoulos* ,† Division of Pharmacology-Pharmacotechnology and Centre of Experimental Surgery, Biomedical Research Foundation of the Academy of Athens, 4 Soranou Efesiou Street, Athens 11527, Greece Received February 17, 2009 The incretin hormone Glucose-dependent Insulinotropic Polypeptide GIP 1-42 (∼5 kDa), is released postprandially, and rapidly degraded by Dipeptidyl Peptidase IV (DP-IV) to yield the inactive GIP 3-42 . Methods for the quantification of the pair of GIP peptides include combinations of immunoassays; however, mass spectrometry based approaches can offer the improved selectivity required for the distinction between the active and inactive forms. In this study, we report an LC/ESI-MS/MS approach for the simultaneous absolute quantification of GIP 1-42 and GIP 3-42 via the corresponding surrogate proteolytic peptide fragments, GIP 1-16 and GIP 3-16 . These surrogate peptides afford approximately 250- fold improvement in lower limits of quantification (LLOQ) compared to the precursor proteins. The LLOQ of the reported method was 5 ng/mL (5-1000 ng/mL) for GIP 1-42 and 10 ng/mL (10-1000 ng/mL) for GIP 3-42 , using 100 µL of mouse plasma. This is the first reported study in which the GIP 1-42 and GIP 3-42 polypeptides are quantified simultaneously with LC/ESI-MS/MS via their tryptic surrogate peptides. The approach is suitable for both preclinical and clinical pharmacokinetic studies due to the low volume required for the analysis. The described methodology was applied to a pharmacokinetic study, in which enhanced stability of exogenously administered GIP 1-42 was demonstrated in mice treated with a DP-IV inhibitor. Keywords: polypeptide absolute quantification • liquid chromatography • tandem mass spectrometry • LC/ESI-MS/MS • glucose-dependent insulinotropic polypeptide • incretin hormones • peptide phar- macokinetics • biomarker verification/validation Introduction Glucose-dependent Insulinotropic Polypeptide GIP 1-42 ,a member of the glucagon peptide superfamily, is a gastric peptide incretin hormone (MW 4983.6). GIP 1-42 is released postprandially and potentiates the glucose-induced insulin response, but is rapidly degraded by Dipeptidyl Peptidase IV (DP-IV) to yield the inactive GIP 3-42 . 1 DP-IV is the major enzyme responsible for the catabolism of the active peptide GIP 1-42 and it cleaves dipeptides from the N-terminus of its substrates. 2 DP-IV is a well-validated target for the treatment of type II diabetes 3 and the incretin hormones, active GIP 1-42 and GLP-1 have been utilized as proximal biomarkers in clinical drug development programs of DP-IV inhibitors such as sitagliptin. 4,5 Exogenously administered incretin hormones GIP 1-42 and GLP-1 and their analogues (e.g., exenatide) have also been studied with respect to their metabolic stability and therapeutic potential. 6-8 Methods for the quantification of the pair of GIP peptides include combinations of immunoassays. Analysis of the active and inactive forms of GIP is an important but difficult task due to low circulating levels 4,9 (50-500 pg/ mL depending on fed or fasted state), rapid clearance and the selectivity required to distinguish between the intact and cleaved protein which differ by only two amino acids. 6,10-13 Similar analytical problems are encountered with other gluca- gon family peptides which are processed with DP-IV such as GLP-1, GLP-2, glucagon, secretin, and growth hormone. 2 Current methodologies toward peptide and protein quanti- fication include immunoassays and mass spectrometric tech- niques. Immunoassays, although sensitive, lack the necessary selectivity for distinction between the peptide, its recombinant forms, metabolites or post-translational modifications. In many cases, suitable antibodies for each protein analyte may not be available. On the other hand, Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) method development is rapid, and applicable to various matrices, thus, providing assays that are suitable for both preclinical and clinical studies, where different species and different tissues need to be analyzed. 14 Tandem mass spectrometry can offer near to absolute struc- tural specificity, analyte detection in low volumes and ease of multiplexing. 15 Despite the recent significant advances in MS methodologies, 16 there are still challenges associated with * To whom correspondence should be addressed: E-mail: ctamvakop@ bioacademy.gr. Fax: +30-210-6597545. Phone: +30-210-6597475. † Division of Pharmacology-Pharmacotechnology. ‡ Centre of Experimental Surgery. 10.1021/pr900155h CCC: $40.75  2009 American Chemical Society Journal of Proteome Research 2009, 8, 3487–3496 3487 Published on Web 05/08/2009