Enantiomeric purity analysis of synthetic peptide therapeutics by direct chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

The determination of chiral purity is critical to the evaluation of the quality of peptide pharmaceutical products. For synthetic peptides, the undesirable D-isomers can be introduced as impurities in amino acid 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 tandem mass spectrometry (HPLC-ESI-MS/MS) method is described that facilitates rapid and accurate determination of amino acid chiral purity of 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 using high-low chromatography and extraction of selected ion response, providing efficiency and simplicity by avoiding the derivatization steps and multiple external standards required by traditional methodologies. GMP method vali-dation feasibility is described for all nineteen chiral proteogenic amino acids. The practical application of the chiral HPLC-ESI-MS/MS method was demonstrated through the recovery of D-amino acid substitutions at each residue of an octapeptide across the 0.1-1.0 % range of interest. The method was applied to the analysis of four model peptides, each consisting of 8-14 amino acid residues, and the results were comparable to those provided by traditional testing methods.

Automated summary

This article describes a chiral high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the rapid and accurate determination of amino acid chiral purity in peptides. The method involves hydrolysis of the peptide in deuterated acid, separation of the amino acids by chiral chromatography interfaced with ESI-MS/MS, and direct analysis of the samples using high-low chromatography and extraction of selected ion response. The method was validated for all nineteen chiral proteogenic amino acids and shown to be comparable to traditional testing methods through the analysis of four model peptides.