Hydrophilic interaction liquid chromatography for dalargin separation from its structural analogues and side products.
Retention behaviour of Dalargin and five peptide analogues of Leu-enkephalin, has been extensively studied by hydrophilic interaction liquid chromatography (HILIC) on a bare silica stationary phase (Atlantis®HILIC silica). The influence of buffer pH, ionic strength, and organic modifier content on peptide retentions was examined. Variation of organic modifier content (70-90% ACN) shows that, as expected, the most polar peptide, Dalargin, is the most retained. Moreover, at acidic pH, the retention mechanism for all the peptides studied seems to rely, mainly, on adsorption phenomenon. By varying the pswH buffer (between 4.4-7.5), we observed that the retention of all the peptides was mainly governed by their total number of charges, whatever the variation (increase or decrease) of their retention factor. At pswH 7.5, an increase of the cationic counter-ion concentration (NH4+) lead to a decrease of the retention factor of Dalargin, suggesting a weak cation exchange for this peptide. For the other peptides, the variation of the retention factors was negligible between 5-15mM. Above 15mM, the retention factors of all the peptides increased, probably due to an increase of the water layer thickness at the surface of the stationary phase. In the second part of the study, qualitative analysis of non-purified dalargin, resulting from solid-phase synthesis, was realized. Optimisation of the separation of the target peptide from its side products has been first performed with UV detection. Then, by coupling the HILIC column with ESI-MS, using the optimal separation conditions, it was possible to identify Dalargin and to propose the amino-acids sequence of its side-products.
Institut Galien Paris-Sud, UMR CNRS 8612, Proteins and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France; Faculty of Pharmacy, Tishreen University, Boulevard Aleppo, Latakia, Syria.
Ayat
A
Abbood
Institut Galien Paris-Sud, UMR CNRS 8612, Proteins and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France.
Claire
C
Smadja
Institut Galien Paris-Sud, UMR CNRS 8612, Proteins and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean Baptiste Clément, 92290 Châtenay-Malabry, France.
Myriam
M
Taverna
LUNAM, University of Nantes, MMS EA 2160, Pharmacy Faculty, 9 rue Bias, F-44035 Nantes, France. Electronic address: claire.smadja@u-psud.fr.
Christine
C
Herrenknecht
D064751:Ammonium Compounds / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004743:Enkephalin, Leucine / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D016308:Enkephalin, Leucine-2-Alanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007477:Ions / Q000737:chemistry; D009994:Osmolar Concentration; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D012822:Silicon Dioxide / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D064751:Ammonium Compounds; D007477:Ions; D010455:Peptides; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D012822:Silicon Dioxide; C040996:enkephalin-Leu, Ala(2)-Arg(6)-
Dalargin side products; Enkephalins; HILIC; Peptides; Silica; Stationary phases