Quantification of arginine-rich cyclic cell-penetrating peptide-lipid conjugates using trifluoracetic acid-based UPLC-MS/MS analysis

Lipid-based drug delivery systems can be surface-modified by lipid conjugates of the substance in question. The most important modifications include arginine-rich cell-penetrating peptides (CPP). A toxicokinetic evaluation of these lipid conjugates is im-portant during preclinical and clinical development of drug delivery formulations. Due to their amphiphilic properties and high number of basic amino acid residues, lipid conjugates of CPP exhibit difficult characteristics in regard to their plasma bioanalysis with LC-MS/MS instruments. These especially include challenging chromatography with extensive carry-over and minimal extrac-tion recovery, and, due to large numbers of basic amino acids and the resulting immobility of protons, resistance against collision-induced dissociation. We developed a surrogate quantification of a CPP-lipid conjugate relying on elimination of the lipid part by phospholipase D digestion. Chromatographic separation was only feasible with trifluoro acetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by post-column addition of aqueous ammonia. Efficient extraction of the surrogate peptide fragment was achieved by protein precipitation with TFA. This enabled the highly sensitive quantification of the CPP-lipid conjugate in plasma in the low picomolar range (lower limit of quantification of 0.1 ng/mL; 34 pM). The assay was validated ac-cording to the pertinent recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the deter-mination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates and especially the TFA-based UPLC-MS/MS analysis with post-column desolvation of TFA adducts by ammonia is a feasible approach for the highly sensitive quantification of arginine-rich peptides and other related substances with challenging chromatographic characteristics.