Synthetic peptide enantiomeric purity analysis by chiral high-performance liquid chromatography-electrospray ionization mass spectrometry

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, D-isomers can be introduced as impurities in amino acid derivative starting materials and can also be formed during peptide synthesis and in some cases during product shelf life. A chiral high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity in a peptide. The peptide is hydrolyzed in deuterated acid to facilitate correction for any racemization occurring during this step of sample preparation, and the amino acids are subsequently separated by chiral chromatography interfaced with ESI-MS/MS for quantitation. The amino acid samples are analyzed directly following hydrolysis by chromatographic separation and extraction of selected ion response. Validation feasibility of the method is described for all nineteen chiral natural amino acids, and the practical application to the analysis of fragments of the synthetic peptide tirzepatide is demonstrated. The method was proven capable of quantitative determination of trace levels of the D-isomer impurities with a reporting limit of 0.1%.