Preparation of novel deuterated cyclosporin A standards for quantitative LC-MS analysis

The investigation on the synthesis of -C deuterated analogues of cyclosporin A (CsA) in N-methylated amino acid residues was performed. The reaction based on our previous report (Bachor et al 2014J Mass Spectrom 49, 43-49) proceeds in the presence of bases like TBD or MTBD at pH13.4. The obtained results suggest the possibility of introduction of 3 deuterons, 2 at the -C of sarcosine, and 1 at the chiral -C atom of 2-N-methyl-(R)-((E)-2-butenyl)-4-methyl-L-threonine residue. The stability of the obtained isotopologues was analyzed both under neutral or acidic conditions, and the obtained results revealed that the introduced deuterons do not undergo back exchange. In addition, the co-elution of deuterated and non-deuterated forms was observed, which opens the possibility of application of the synthesized isotopologues for the quantitative analysis utilizing isotope dilution strategy. The proposed strategy of the deuterated CsA analogues synthesis is rapid, cost-efficient, and does not require derivatization reagents or further purification and may be useful in quantitative analysis of cyclosporin A and its metabolites.