# A Label-Free Liquid Chromatography–Tandem Mass Spectrometry Method for the Quantitative Analysis of Exosome Pharmacokinetics In Vivo

## TL;DR

A new method allows tracking of exosome behavior in the body without altering them, aiding drug development.

## Contribution

A label-free LC-MS/MS method for quantifying exosome pharmacokinetics in vivo is developed.

## Key findings

- The method was validated for specificity, linearity, sensitivity, and reproducibility.
- The PK profile of HEK 293F-derived exosomes in rats was successfully characterized.
- The approach enables direct, label-free quantification of exosomes in plasma.

## Abstract

Background: Exosomes are nanoscale extracellular vesicles actively secreted by cells that play critical roles in disease biomarker discovery, drug delivery, and direct therapeutic applications. However, in vivo pharmacokinetic (PK) studies of exosomes remain limited, hindering their clinical translation. Due to their complex and heterogeneous composition, most existing PK methods rely on chemical or genetic labeling, which may alter their native behavior and complicate accurate analysis. Methods: To address this challenge, we developed a label-free liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to investigate the PK of naive exosome-based therapeutic modalities. Exosomes were isolated from rat plasma using size exclusion chromatography (SEC) and quantified using multiple reaction monitoring (MRM) targeting specific exosomal peptides as surrogate analytes. Following intravenous administration of exosomes via the tail vein, plasma concentrations were determined by peptide peak areas, and PK parameters were calculated using a non-compartmental model. Results: The method was rigorously validated for specificity, linearity, sensitivity, and reproducibility. Its feasibility was further confirmed by successfully characterizing the PK profile of HEK 293F-derived exosomes in rats. Conclusions: This analytical strategy enables direct, label-free quantification of exosomes in plasma and provides a robust platform for advancing exosome-based drug development and translational research.