Advanced 2D-HPLC–MS/MS Approach for Cosmetic Peptide Analysis in Skin Homogenates

Peptides are increasingly employed as active ingredients in both therapeutic and cosmeceutical applications due to their high biological specificity, favorable safety profiles, and expanding market relevance. In topical formulations, however, their efficacy is strongly influenced by stability within the dermal environment, where widespread proteolytic enzymes can promote degradation and limit bioavailability. Despite the growing use of bioactive peptides in dermatology and cosmetics, robust analytical methodologies for assessing their resistance to enzymatic degradation in biologically relevant matrices remain limited. In this study, a two-dimensional (2D) HPLC–MS/MS method based on ion trap detection was developed and validated for the quantitative evaluation of peptide stability in human skin homogenate (HSH). The analytical platform integrates on-line sample clean-up with chromatographic separation on a silica-based pentafluorophenyl (PFP) column, enabling reliable analysis of peptides with markedly different polarity profiles within a single workflow. The method supports direct injection of samples containing a relatively high organic solvent content, allowing limited sample dilution, reducing sample handling, and preserving analytical sensitivity. The loading step was optimized to ensure efficient trapping of highly hydrophilic peptides, while minimizing matrix-related interferences. A key feature of the method is the use of isomeric peptide analogues (scrambled and reverse-sequence peptides) as internal standards. These compounds share identical precursor ions with their corresponding analytes and can be monitored under the same MS/MS conditions, ensuring effective correction for variability in ionization and fragmentation. This strategy represents a practical and scalable alternative to stable isotope-labelled standards, particularly in exploratory or screening studies. The ion trap mass analyzer further contributed to method robustness by providing controlled and reproducible fragmentation behavior with cosistent product ion distributions. The validated method demonstrated satisfactory linearity, sensitivity, precision, accuracy, and minimal matrix effects. Its applicability was confirmed through stability studies of peptides with distinct polarity characteristics in HSH, highlighting different degradation behaviors under enzymatic conditions. Overall, the proposed 2D-HPLC–MS/MS approach provides a versatile analytical platform for peptide stability assessment in complex dermal matrices and supports early-stage screening and preclinical evaluation of bioactive peptides intended for topical applications.