Analysis of native amino acid and peptide enantiomers by high‐performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

Abstract

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry was used for the separation and detection of amino acid and peptide enantiomers. With detection limits as low as 250 pg, 25 amino acids enantiomers were baseline resolved on a Chirobiotic T chiral stationary phase. APCI demonstrated an order of magnitude better sensitivity over electrospray ionization (ESI) for free amino acids and low molecular mass peptides at the high LC flow-rates necessary for rapid analysis. As the peptide chain length increased (peptides with M(r) > or = 300 Da), however, ESI proved to be the more ideal atmospheric pressure ionization source. A mobile phase consisting of 1% (w/w) ammonium trifluoroacetate in methanol and 0.1% (w/w) formic acid in water increased the sensitivity of the APCI method significantly. A step gradient was then used to separate simultaneously all 19 native protein amino acid enantiomers in less than 20 min using extracted ion chromatograms.

Could linear solvation energy relationships give insights into chiral recognition mechanisms?

“…They have exceptional selectivity for amino acids [5,6], and have proven to be useful for the enantiomeric separation of a wide variety of molecules [7]. Chemically, they are composed of a multiplicity of functional groups, including hydroxyls, phenols, amides, aromatic rings, various carbohydrate moieties, ionizable carboxylic acid and amine groups, as well as a basket shaped structure [8].…”

Considerations on HILIC and polar organic solvent‐based separations: Use of cyclodextrin and macrocyclic glycopetide stationary phases

“…Different ionization methods were compared: APCI-MS gave best sensitivity for small analytes under M r 200, ESI-MS worked best for large peptides with M r larger than 300, and similar performance between APCI and ESI was observed for M r 200 -300 analytes [71]. The addition of mobile phase additives, ammonium trifluoroacetate, and formic acid, could increase the sensitivity by an order of magnitude for amino acids [71]. Detection limits for amino acids were comparable between APCI and ESI, and the lowest reported detection limit was 250 pg [71].…”

“…LC-MS is the most desirable detection method for this analyte -mobile phase combination. Moreover, MS can provide a second dimension separation and increased peak capacity for complicated compound mixtures [71]. Different ionization methods were compared: APCI-MS gave best sensitivity for small analytes under M r 200, ESI-MS worked best for large peptides with M r larger than 300, and similar performance between APCI and ESI was observed for M r 200 -300 analytes [71].…”

“…The addition of mobile phase additives, ammonium trifluoroacetate, and formic acid, could increase the sensitivity by an order of magnitude for amino acids [71]. Detection limits for amino acids were comparable between APCI and ESI, and the lowest reported detection limit was 250 pg [71]. ESI-MS provided at least two orders of magnitude lower detection limit for large peptides than UV 210 nm detection; an LOD of 2 ng was reported for Vasopressin peptides (9 amino acid residues) using the Chirobiotic TAG column [83].…”

“…Moreover, MS can provide a second dimension separation and increased peak capacity for complicated compound mixtures [71]. Different ionization methods were compared: APCI-MS gave best sensitivity for small analytes under M r 200, ESI-MS worked best for large peptides with M r larger than 300, and similar performance between APCI and ESI was observed for M r 200 -300 analytes [71]. The addition of mobile phase additives, ammonium trifluoroacetate, and formic acid, could increase the sensitivity by an order of magnitude for amino acids [71].…”