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 pertinent substance. Prominent modifica-tions include arginine-rich cell-penetrating peptides (CPP). Toxicokinetic evaluation of these lipid-conjugates is important during pre- and clinical development of drug-delivery formulations. Due to their amphiphilic character and high number of basic amino acid residues, lipid-conjugates of CPP exhibit challenging characteristics in regard to their plasma bioanalysis with LC-MS/MS instruments. These especially include challenging chromatography and minimal extraction recovery, and, due to large numbers of basic amino acids and the resulting immobility of protons, resistance against collision-induced dissociation. We developed a surro-gate quantification of a CPP-lipid-conjugate relying on elimination of the lipid-part by phospholipase D digestion. Chromatograph-ic 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 according to the pertinent guidelines of the FDA and EMA on bioanalytical method validation and applied to the determination of 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 for arginine rich peptides and other substances with challenging chromatographic characteristics.