A simple and rapid LC-MS/MS and CE-MS/MS analytical strategy for the determination of therapeutic peptides in modern immunotherapeutics and biopharmaceutics

[Display omitted] •Modern and progressive UHPLC-MS/MS and CE-MS/MS analysis of peptide conjugates.•Fast non-enzymatic peptide cleavage.•Sensitive and selective detection of immunogenic peptides with minimum sample pretreatment.•The promising effective and specific analytical method for characterization of biologics. Modern therapy of metabolic, neurodegenerative, inflammation, or cancer diseases is recently based on an immunotherapeutic approach. The peptide conjugates represent innovative and effective therapeutics that are better tolerated and are much more specific than small molecule-based medicines. The nature and manufacturing process of these therapeutics make their analysis very challenging. Here, two robust analytical methods based on an on-line combination of ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) and capillary electrophoresis with tandem mass spectrometry (CE-MS/MS) were developed for fast determination of immunogenic synthetic peptide (peptide sequence CADNLHKVVGQST) in a conjugate with bovine serum albumin (BSA) as a carrier protein and is a peptide, conjugate formulated with a vaccine adjuvant – Alhydrogel® 2 %. An effective non-enzymatic release step of the peptide from the final peptide conjugate based on acid hydrolysis with the use of 2% formic acid was successfully tested and implemented. The proposed methods were validated according to the ICH guideline and parameters such as linearity, precision, and accuracy, the limit of detection (LOD) or limit of quantification (LOQ) were assessed. Calibration curves were linear within the range of 1–30 μg.mL−1 and the correlation coefficients were higher than 0.99. The intraday and interday precisions were 3.2–8.1 % (UHPLC-MS/MS), 1.6–9.3 % (CE-MS/MS) and 3.6–10.3 % (UHPLC-MS/MS), 4.1–10.2 % (CE-MS/MS), respectively. The recovery ranged in the interval of 98.4–107.4 % for UHPLC-MS/MS method and 100.3–103.2 % for CE-MS/MS method. The presented approaches represent an effective tool for simple, rapid and robust quantification of immunogens in modern immunotherapeutics and other biopharmaceuticals with appropriate peptide sequences.