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Introduction and Objectives More attentions were paid to RT(retention time)information in label-free LC-MS/MS quantification.Real RT can be obtained in an enough elution time,which offers exchange equilibrium in column,while there were few study focused on it.ACN concentration of peptide RT was figured out in different elution time in this study,and an appropriate elution method for peptide separate was displayed.Methods: Dead time of column(Thermo 75 μm × 50 cm,2 μm,100 A,C18 packing material)was measured with urasil using 80%ACN and 20%water solvent.Gradient delay time of liquid chromatography(Eksigent 425 system)was measured with a TUV detector(Waters)at 265 nm,using a linear gradient from 100%solvent A(100%water)and 0%solvent B(10%acetone,90%water)to 25%solvent B over 30,60 and 90 min.RTs of 12 peptides from Pierce Retention Time Calibration Mixture(Thermo)were collected from 95%solvent A(100%water,0.1%formic acid)and 5%solvent B(100%ACN,0.1%formic acid)to 35%solvent B over different times.Results and Discussion Research PeakView(AB SCIEX)was applied to peptide RT detection,and with the data of column dead time and gradient delay time,ACN concentration of peptide RT can be calculated accurately.A figure was plotted,which showed ACN concentration of peptides to elution time.And the best linear gradient of 0.25%ACN/min was found,which showed stable ACN concentration.The gradient of 0.167%ACN/min could also offer a stable ACN concentration of RT,while it lead to a wider peak width.Conclusions Since the 0.25%ACN/min linear gradient method predicted retention time in a coefficient of 0.9844 with the Sequence Specific Retention Calculator,our study showed an appropriate elution gradient for peptide separate in RPLC.