Impurity identification and determination for the peptide hormone angiotensin I by liquid chromatography–high-resolution tandem mass spectrometry and the metrological impact on value assignments by amino acid analysis

It is common practice to quantify the mass concentration of a peptide solution through quantitative determination of selected chemically stable amino acids produced following complete hydrolysis of the parent peptide. This is because there is generally an insufficient quantity of material available to allow for the obvious alternative of a direct purity analysis characterization of the parent peptide, and the subsequent constitution of a calibration solution. However, selected accurately characterized pure peptide reference materials are required to establish reference points for the dissemination of metrologically traceable measurements and to develop reference measurement systems for laboratory medicine. In principle, purity assignment of a peptide can be performed by using the so-called mass balance approach, by employing a range of analytical techniques to obtain an estimate of the mass fraction content of all impurities present in the intact peptide, and by utilizing the difference from the theoretical limit value to assign the mass fraction content of the main peptide. Liquid chromatography–high-resolution tandem mass spectrometry (LC-hrMS/MS) is a key technique for the detection, identification, and determination of structurally related impurities present in a peptide material, and experiments characterizing the model peptide hormone angiotensin I (ANG I) are described in the present work. Degradation products that were generated from ANG I after storage at elevated temperatures were screened. The formation of peptide fragments such as ANG II or ANG III was determined by comparison of measured mass values with calculated mass values. The use of a data-dependent acquisition technique enabled the detection and structural characterization of ANG II and other peptide fragments as major impurities in the same LC-hrMS/MS analysis run. Subsequent quantification using external calibration allowed the mass fraction of the major impurities in a candidate reference material to be estimated as 10.4 mg/g. Failure to correct for these impurities would lead to a 1 % error in the determination of the concentration of the peptide in solution by amino acid analysis techniques.