Preliminary results for the development of a peptide screening method by means of LC-MS / MS

Bioactive peptides such as insulins, synthetic adrenocorticotrophic hormone (ACTH) analogue Synacthen, Gonadorelin (LHRH) and insulin-like growth factors (IGF) provide a reasonable potential for the misuse as performance enhancing agents and are prohibited in elite sports according to the list of banned substances established by the WADA. Currently, determination of these target analytes is possible by single assays only. The present method provides results for a preliminary approach to determine various prohibited peptides occurring in urine (e.g. Gonadorelin, Humalog (Insulin Lispro), Apidra (Insulin Glulisine), Novolog (Insulin Aspart), Lantus (Insulin Glargine), Porcine Insulin, Bovine Insulin, IGF-1 etc.) in one screening procedure. The method enables the effective, highly sensitive and specific screening for several different target analytes that are simultaneously purified and analysed by means of immunoaffinity purification, subsequent liquid-chromatographic separation and high resolution / high accuracy mass spectrometric determination. Principally, the approach is extendable to any banned peptide, if adequate antibodies are available. At the present status of the project only a limited number of analytes were implemented in the method. Introduction The analysis of performance enhancing peptides or small proteins has reached an established status in the sports drug testing program of many doping control laboratories. Recently, a considerable number of methods to uncover the frequently reported misuse of peptides were published, and single assays were implemented into routine doping controls (1-18). Unfortunately, each class of these bioactive compounds (e.g. synthetic insulins, gonadorelin, Synacthen, IGF-1 etc.) requires a dedicated sample preparation procedure and, thus, the WS2010 LECTURE 109 workflow is, in comparison to commonly used screening methods for small molecules (e.g. stimulants, anabolic agents etc.) and less effective. A simple combination of the different assays was hindered due to the heterogeneous character of the target analytes on the one hand and the low concentrations (in low fmol/mL) in urine on the other hand. Usually these challenges were handled by a highly specific and effective purification step using immunoaffinity approaches and additionally an enhanced chromatographic and mass spectrometric detection system composed of nanoscale UPLC coupled nano-electrospray ionisation and tandem mass spectrometry (6, 8, 9, 19). The present study provides preliminary results for a combination of different assays resulting in a screening method for various different peptides without losing the necessary sensitivity or specificity. The approach is based on the simultaneous usage of different primary antibodies for the immunoaffinity step and combined purification with secondary antibody coated magnetic beads. Generally, the method is not limited to the presented analytes and expandable to further peptides or proteins if appropriate antibodies (AB) are available. Chemicals and Reagents Water, acetonitrile, trifluoroacetic acid and formic acid (all ultrapure for nano-liquid chromatography) were purchased from Biosolve (Valkenswaard, The Netherlands). Acetic acid (glacial), acetonitrile (analytical grade), sodium dihydrogenphosphate dihydrate (p.a.), disodium hydrogenphosphate dodecahydrate (p.a.), and sodium chloride (p.a.) were purchased from Merck (Darmstadt, Germany). Tris(carboxyethyl)phosphine hydrochloride (TCEP-HCl) was from Sigma (Deisendorf, Germany) and coated Dynal beads (anti-rabbit IgG, anti-mouse IgG) were obtained from Invitrogen (Karlsruhe, Germany). Polyclonal anti-ACTH antibodies (serum, anti-rabbit) and Anti-LHRH AB (serum, anti-rabbit) were purchased from Acris antibodies (Herford, Germany) and monoclonal Anti-Insulin AB (ascide fluid, anti mouse) and Anti-IGF-1 AB (polyclonal, Host: rabbit) were obtained from CER-groupe (Marloie, Belgium). Insulin analogues Humalog, Novolog, Apidra and Lantus were supplied by Eli Lilly (Indianapolis, IN), Novo Nordisk (Princeton, NJ), and Aventis (Kansas City, MO). Porcine insulin and bovine insulin were from Sigma (Deisendorf, Germany). LHRH reference substance was supplied as pharmaceutical formulation Kryptocur® by Sanofi-Aventis (Frankfurt, Germany). Synacthen Depot 1 mg was from Novartis Pharma (Bern, Switzerland). N-Acetyl-ACTH Fragment 1-17 used as internal standard (IS) was from Bachem (Bubendorf, Switzerland). Solid phase extraction cartridges OASIS HLB (60 mg, 3 mL) were bought from Waters (Eschborn, Germany). IGF-1, longR-IGF-1, R-IGF-1 and Des1-3-IGF-1 were WS2010 LECTURE 110 obtained from IBT-Biosystems (Reutlingen, Germany). All aqueous buffers and solutions were prepared in MilliQ water. Sample preparation The sample preparation procedure is schematically described in Fig. 1 and was described in detail earlier (19). Due to the fact that the presented data is preliminary and a final characterization and optimization of critical parameters are not finished yet, the detailed description will follow, but the main steps of the method will consist of solid phase extraction of 5 mL of urine, magnetic bead based immunoaffinity purification and subsequent LCMS/MS detection. Details were also described earlier. (8, 9, 19) Figure 1: Scheme of the sample preparation procedure 5 mL urine + ISTDs