INTRODUCTION Chemical quality control of peptide functionality testing in tissue baths. M. Verbeken 1, S. Van Dorpe 1, R. Lefebvre 2 and B. De Spiegeleer 1* 1 DruQuaR, Harelbekestraat 72, B-9000 Ghent, Belgium 2 Departement of Farmacology, De Pintelaan 185, 9000 Ghent * Corresponding author: (Ref.: EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSIONS DruQuaR [1] Tirapelli CR, Bonaventura D, Tirapelli LF, et al.(2009). Mechanisms underlying the vascular actions of Endothelin 1, Angiotensin II and Bradykinin in the rat carotic. Pharmacology, 84 (2): REFERENCES Tissue-bath experiments Guinea pig ileum (gpI) smooth muscle preparations were mounted using cotton threads in glass tissue bath units. The units were filled with Krebs buffer, maintained at 37°C. Equilibration and electrical field stimulation through platinum electrodes preceded model peptide administration. The model peptides (10 -5 M) were subjected for ten minutes to the tissue bath environment (TB treatment). A tissue bath sample (1 ml) was collected and used for analytical testing. Typical results are given in Table 1. Tissue bath (TB) samples (10 min in TB medium with gpI strips) were compared with control samples (no TB treatment).The reporting treshold (RT) used, was defined as 1% area of the main peptide peak in the control samples. Peak purity at 195 nm (normalization), the number of peaks with a RT > 1% and the relative area concentration of the highest impurity of the control samples are listed as well. The peptides remaining in TB samples are assayed, relative to the corresponding peptides in the control sample (recovery), accepted as standard. Table 1: Chemical quality control of model peptides using tissue baths (UV 195 nm) The behaviour of peptides in tissue bath experiments has lead to the partitioning of peptides in two major groups: (1) stable peptides: no adsorption or degradation (chemical as well as metabolic) (2) unstable peptides: loss of peptide occurs by: adsorption without degradation; or degradation without adsorption; or adsorption and degradation Overall, our findings call for a general quality control system when functionality of peptides is studied in tissue bath experiments. Figure 2. HPLC-chromatogram of control (left) and TB (right) sample: SBO 379 peptide peak with areas (MA). Tissue baths are used in the study of functional effects of compounds, including peptides, which can lead to the development of new pharmaceuticals [1]. However, peptides present special characteristics which make tissue bath experiments challenging: adsorption of peptides to contacting materials, metabolisation of peptides by the tissue and/or chemical degradation during the experiments. The chemical integrity of the peptides is most often not verified during these tissue bath experiments, thereby blurring the final functionality conclusions. Chemical quality control of peptides The chemical quality control was based upon the comparison of the chemical quality of the model peptide control solution with the tissue bath sample solution using HPLC. A C18 reversed phase column with gradient program (acetonitril/water mixture) and UV detection was used for quantification, while HPLC-ESI/iontrap MS n was used for identification purposes. SBO 33_11SBO 112SBO 121SBO 215_7SBO 318SBO 397 Control samples Peptide purity 73.3 %69.0 %93.6 %97.4 %100.0 %100 % n impurity peaks Level max impurity 26.7 %31.0 %6.4 %2.6 %N/A TB samples Recovery main peak22.7 %5.2 %8.0 %26.3 %20.0 %92.3 % n impurity peaks Level max impurity 16.7 %25.0 %27.3 %71.4 %24.0 %N/A Mass balance 20 %4.8 %23.4 %89.7 %83.3 %92.3 % ObservationsAdsorption Adsorption/ Degradation Stable peptide MS UV N/A: not applicable, as only one peak is observed due to the main peak present MS UV Figure 1. HPLC UV-MS spectrum for a model peptide MA: 0.39 MA: 0.36