Peptide-based chiral derivatizing reagents in nano-scale liquid chromatography: Effect of the oxidation state of cysteine moiety on enantioseparation of ibuprofen

Mass spectrometry is a powerful instrument for identifying the structures of target compounds. However, one of the major disadvantages is that mass spectrometry could not directly distinguish between enantiomers. Cysteine, a special amino acid containing a thiol group, can undergo a variety of oxidative modifications. The different redox states of cysteine existing in a peptide or protein may interact differently with the stationary phase in a chromatographic column. In this study, we developed a peptide that can be used as a chiral derivatization reagent for the enantioseparation of drugs using nano-scale liquid chromatography. Ibuprofen, a model analyte to evaluate the chiral separation efficiency of peptide-based derivatizing reagents, is a widely used antiinflammatory drug. After the peptide derivatizing reagents were micro-coupled with ibuprofen, the solution was injected into a nano-flow column. The separation of the (R) and (S) forms of ibuprofen was performed on the nano-scale, using a C18 75 mu m capillary column at a flow rate of 400 nL/min. The results indicated that the peptide AAAACAAAR comprising tri-oxidized cysteine is most suitable for separating (R)- and (S)-ibuprofen at the nano-scale flow rate without using a chiral column. The detection of (R)- and (S)-ibuprofen in only 10 mu L human plasma (by pricking the finger) was sufficient with an injection volume of 200 nL. The linear range for detecting the (R) and (S) forms of ibuprofen in plasma was 1-100 mu g/mL, the relative standard deviation (RSD) for the precision analyses was below 17 %, and the recovery for the method was 86%-109%. Using the peptide as a chiral derivatization reagent with a suitable cysteine oxidation state, we successfully separated and quantitated the (R) and (S) forms of ibuprofen in commercial tablets and plasma samples. Finally, our computation simulations suggested that the oxidization state of cysteine affects the formation of solute-solvent hydrogen bonds, which might influence the separation efficiency on the C18 column by changing the molecular binding affinity with the column stationary phase.

Automated summary

This study developed a peptide-based chiral derivatization reagent for the successful separation and quantitation of the (R) and (S) forms of ibuprofen in nano-scale liquid chromatography. Using a suitable cysteine oxidation state, the peptide as a chiral derivatization reagent enabled the separation of enantiomers in commercial tablets and plasma samples. Computational simulations also suggested that the oxidation state of cysteine affects solute-solvent hydrogen bond formation, influencing the separation efficiency on the C18 column.