Quantification of Arginine-Rich Cyclic Cell-Penetrating Peptide-Lipid Conjugates Using a Surrogate Peptide Extracted...

For the toxicokinetic evaluation of a lipid conjugate of the cell-penetrating peptide cyclic nonaarginine, we aimed to develop a highly sensitive plasma quantification. Due to the amphiphilic properties and high number of basic amino acids, plasma bioanalysis with LC-MS/MS instruments is difficult. Challenges in particular include chromatographic characteristics, minimal extraction recovery, and resistance against collision-induced dissociation. We developed a quantification of a nonextractable CPP-lipid conjugate by eliminating the lipid part by phospholipase D digestion, enabling the efficient extraction of a surrogate peptide for analysis. Chromatographic separation was feasible only with trifluoroacetic acid (TFA)-based mobile phases. Ion suppression caused by TFA was reversed by postcolumn 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 recommendations of the ICH M10 guideline on bioanalytical method validation and applied to the determination of the intravenous pharmacokinetics of the CPP-lipid conjugate in Beagle dogs. Applicability was further demonstrated by validation of the assay for human plasma. The established strategy can be used as a general approach to the bioanalysis of amphiphilic lipid conjugates by using lipase digestion to generate extractable surrogate fragments. Additionally, TFA-based UPLC-MS/MS analysis with desolvation of TFA adducts by postcolumn addition of ammonia is feasible for the regulated, highly sensitive quantification of arginine-rich peptides.