LC-MS/MS Validation Analysis of Trastuzumab Using dSIL Approach for Evaluating Pharmacokinetics.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Quantitative targeted proteomics using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is emerging as a powerful alternative to ligand-binding assays.
• Advancements in mass spectrometry enhance specificity, selectivity, sensitivity, and eliminate the need for antibody generation, broadening clinical applications.

Purpose of the Study:

• To develop and validate a novel double Stable Isotope Label (dSIL) quantitative proteomics assay for Herclon® (trastuzumab).
• To assess proteolytic digestion efficiency and accurately quantify Herclon® in human serum using signature peptides from its complementary determining region (CDR).

Main Methods:

• A double Stable Isotope Label (dSIL) approach was employed using two distinct surrogate peptides.
• Quantitative analysis was performed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
• Method validation was conducted according to established bioanalytical guidelines.

Main Results:

• The dSIL LC-MS/MS assay demonstrated good linearity for Herclon® quantification over a range of 5-500 µg/mL.
• Validation data confirmed compliance with bioanalytical regulatory guidelines.
• The method effectively evaluated proteolytic digestion efficiency and analyte quantification.

Conclusions:

• The developed dSIL LC-MS/MS assay provides a robust and accurate method for quantifying Herclon® in human serum.
• This quantitative proteomics approach offers a validated, antibody-free alternative for Herclon® bioanalysis.
• The findings support the utility of advanced mass spectrometry techniques in clinical applications.