1. INTRODUCTION Concentration-dependent peptide losses during the drying step in the sample preparation: influence of additives Adel Pezeshki 1,2, Valentijn Vergote 1, Christian Burvenich 2, Bart De Spiegeleer 1 * 1 Drug Quality & Registration (DruQuaR) group, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium, 2 Department of Physiology and Biometrics, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. *Corresponding author: bart.despiegeleer@ugent.be (O.Ref.: 2009–221c)bart.despiegeleer@ugent.be EXPERIMENTAL RESULTS AND DISCUSSION CONCLUSIONS DruQuaR Table 1: Peptide HPLC method Column Vydac Everest RP-C18 250 mm × 4.6 mm, 5 µm, 300 Å Column temperature30 ° C Flow rate1 ml/min Mobile phase constituents A = Water with 0.1% m/V formic acid B = Acetonitrile with 0.1% m/V formic acid Gradient Isocratic for 1 min: 90% A and 10%B Linear gradient to 40% B at 60 min Sample temperatureAmbient Injection volume30 µL DetectionUV @ 275 nm PeptideAdditive Recovery (%) SpeedVacLyoVac PlasticGlassPlasticGlass Leucine-enkephalin Mannitol90.3 ± 3.598.9 ± 4.289.0 ± 2.994.6 ± 3.4 Water97.6 ± 9.197.2 ± 8.393.2 ± 7.496.2 ± 9.1 DMSO96.5 ± 5.593.2 ± 4.785.1 ± 4.791.4 ± 3.4 DMF89.8 ± 3.5101.9 ± 1.090.9 ± 1.693.3 ± 3.3 PEG 40089.4 ± 3.3 97.8 ± 3.683.4 ± 5.293.3 ± 3.1 n-β-D-Glucopyranoside95.9 ± 1.6100.9 ± 2.991.4 ± 2.095.0 ± 1.4 Goserelin Mannitol92.3 ± 4.692.3 ± 6.799.2 ± 3.164.3 ± 3.6 Water84.4 ± 4.5102.2 ± 8.8100.2 ± 4.773.5 ± 5.3 DMSO 88.8 ± 5.1100.6 ± 5.796.1 ± 5.572.1 ± 4.5 DMF89.7 ± 6.890.8 ± 7.292.4 ± 5.872.8 ± 6.5 PEG 40085.7 ± 8.292.8 ± 7.871.4 ± 6.654.0 ± 7.7 n-β-D-Glucopyranoside93.1 ± 1.392.9 ± 8.390.4 ± 2.590.7 ± 2.5 Bovine insulin Mannitol62.8 ± 0.883.6 ± 4.2 81.7 ± 4.094.7 ± 5.3 Water82.4 ± 5.183.9 ± 6.090.3 ± 6.194.8 ± 2.4 DMSO92.6 ± 7.192.5 ± 7.494.1 ± 8.4103.6 ± 9.2 DMF74.1 ± 2.789.2 ± 4.591.8 ± 5.198.6 ± 3.8 PEG 40095.8 ± 5.581.0 ± 5.264.7 ± 2.195.7 ± 3.3 n-β-D-Glucopyranoside95.4 ± 7.192.1 ± 6.789.3 ± 5.088.9 ± 4.9 Buserelin Mannitol91.9 ± 4.493.4 ± 1.485.0 ± 3.171.9 ± 0.9 Water86.4 ± 3.891.6 ± 8.689.5 ± 1.582.4 ± 2.7 DMSO86.4 ± 1.893.4 ± 1.2103.7 ± 2.181.7 ± 2.1 DMF91.1 ± 2.490.1 ± 1.294.2 ± 6.777.7 ± 0.9 PEG 40079.0 ± 2.2105.7 ± 3.3 94.1 ± 2.479.2 ± 5.0 n-β-D-Glucopyranoside96.2 ± 1.792.0 ± 3.892.0 ± 2.495.1 ± 1.7 Mouse obestatin Mannitol95.3 ± 3.1 93.7 ± 4.490.8 ± 6.095.2 ± 4.7 Water90.8 ± 2.891.1 ± 11.091.2 ± 3.088.5 ± 4.0 DMSO99.8 ± 3.791.2 ± 6.195.8 ± 4.091.1 ± 2.7 DMF101.4 ± 7.8 90.2 ± 7.4101.3 ± 8.091.8 ± 7.0 PEG 400101.8 ± 8.3 93.2 ± 3.482.7 ± 4.487.2 ± 1.5 n-β-D-Glucopyranoside97.2 ± 2.6102.5 ± 3.692.6 ± 2.896.1 ± 2.3 Table 2 Peptide loss during sample evaporation It is expected that bovine teat canal, teat cistern and/or mammary epithelial cells produce antimicrobial peptides as the first line of defense against pathogens, especially during dry period. Peptide profiling of udder tissues will give us more information about the possible roles of peptides (peptidomics). However, variable recovery of peptides during sample preparation is a well-known phenomenon. Peptide loss, mainly due to adsorption, can occur during sample treatment [1]. Removal of the solvent from samples by drying (both centrifugal evaporation and freeze-drying) was found to significantly reduce the recovery of model peptides [1]. Efforts to reduce this adsorption loss by organic additives is only scarcely elaborated until now. The objective of this study was to investigate the influence of five different additives in order to minimize peptide loss during sample preparation. E XPERIMENT 1: E FFECT OF ADDITIVES ON MODEL PEPTIDES RECOVERY A mixture of model peptides (0.2 µg/µl each), i.e. bovine insulin, buserelin, goserelin, leucine-enkephalin, and mouse obestatin prepared by water/acetonitrile mixture (95:5 V/V) was pipetted into total recovery glass vials containing the five separate additives, i.e. DMSO, DMF, PEG 400, mannitol (0.5% W/V aqueous solution) and n-nonyl-β-D- glucopyranoside (0.05% W/V aqueous solution). 95 µl of model peptides mixture was added to vials containing the five µl of selected additives. The samples were analyzed by HPLC (Table 1). E XPERIMENT 2: E FFECT OF ADDITIVES ON MODEL PEPTIDE RECOVERY INCLUDING DRYING AND VIAL MATERIAL EFFECTS A 0.2 µg/µl mixture of model peptides was prepared in water. Five µl of additives were pipetted into vials made of polypropylene and glass containing 95 µl of the model peptides mixture. After drying the samples by centrifugal evaporation or freeze-drying, 95 µl of water/acetonitrile mixture (95:5 V/V) was added to the samples (recovery values were corrected for water-drying loss in case of mannitol and n-nonyl-β-D-glucopyranoside aqueous solutions, water, DMSO, and DMF). Samples were analyzed by HPLC (Table 1). E XPERIMENT 3: I NFLUENCE OF PEPTIDE CONCENTRATION Different concentrations (200, 100, 20 and 2 µg/ml each) of model peptides mixtures were prepared in ACN/water (5/95%, v/v). These model peptide mixtures were prepared in polypropylene vials and dried by Speedvac. The residue was re-dissolved in ACN/water (5/95%, v/v) and the resulting samples were analyzed by HPLC (Table 1).  No significant effect of the additive presence on the quantitative chromatography was demonstrated.  The overall recovery values of model peptides after the drying step using different organic additives ranged from 86% to almost 94% (i.e. when mannitol and n-nonyl- β-D-glucopyranoside is used, respectively)(Table 2).  n-nonyl-β-D-Glucopyranoside is the best additive for recovery of peptides during centrifugal evaporation as well as during freeze-drying (Table 2).  n-nonyl-β-D-Glucopyranoside not only showed the highest recovery, but also gave more robust results, i.e. less influenced by the operational variables (Table 2).  As expected, at lower concentrations, higher loss of peptides is observed which can be adequately described by the Freundlich equation (R 2 ≥0.999). (Figure 1). Applying n-nonyl-β-D-glucopyranoside as an additive can prevent or decrease peptide loss during the selected solvent removal evaporation procedure. The peptide concentration effect of our study can be adequately described by the Freundlich equation. REFERENCES 1 Pezeshki, A., V. Vergote, S. Van Dorpe, B. Baert, C. Burvenich, A. Popkov, B. De Spiegeleer. Journal of Pharmaceutical and Biomedical Analysis. 49 (2009) 607–612. ACKNOWLEDGEMENTS: We would like to thank Koen Depoorter for his operational help in the experiments. Drug Quality & Registration (DruQuaR) format Man06.001.006-v01 Figure 1 Effect of concenration on model peptides adsorpation to container surfaces