Accurate quantification of synthetic peptides by amino acid-stable isotope dilution mass spectrometry

Abstract

An approach for quantification of peptides by hydrolysis followed by reversed-phase liquid chromatography (RPLC)-stable isotope dilution mass spectrometry (MS) is presented. The purities of all the determined peptides were greater than 99% by using RPLC and over 90% detected by MS, indicating that the method was workable. The hydrolysis conditions optimized were 4-6 h with 6 mol/L HCl at 150 degrees C. Two or more amino acids were chosen to ensure the accuracy of the determined results. The content range of peptides was determined to be 62.07% - 88.18% with the relative standard deviations less than 8% and relative errors less than 5%. Besides Phe, Val and Ile which were commonly used for the analysis of peptide contents, Arg as another amino acid for peptide quantification can be chosen to enhance the popularity of the method. In a word, application of this method for direct determination of peptide content can avoid the side effects in the derivatization of amino acids and the tedious operation in liquid chromatography. This will improve the precision and accuracy of the method and provide an alternative for peptide quantification.