# Capillary Electrophoresis as a Useful Tool to Separate Isomeric Opioid–Neurotensin Hybrid Peptides

## TL;DR

This paper describes a capillary electrophoresis method to separate two opioid-neurotensin hybrid peptides that differ by a single amino acid.

## Contribution

A validated CE method is introduced for resolving isomeric opioid–neurotensin hybrids with high precision and speed.

## Key findings

- The CE method achieved baseline resolution (Rs = 1.4) for the two isomeric peptides.
- The method demonstrated excellent linearity (R² 0.9991–0.9999) and recovery rates (94.8–100%).
- The analysis time was only 6 minutes, and the method works in both aqueous and serum samples.

## Abstract

We developed and validated a capillary electrophoresis (CE) method for the separation of two opioid–neurotensin hybrid peptides, recently presented as potent analgesics being decapeptides with a hybridic nature (i.e., H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH; PK20 and its structural analogue H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Ile-Leu-OH; [Ile9]PK20). As these two chimeras differ by only one amino acid, Tle→Ile, and are characterized by possessing the same molecular weight while having different spatial conformations, the aim of the study was to determine their potential separation in terms of the presence of any differences resulting from this structural modification. The separation process was performed using an eCAP fused silica capillary at a detection wavelength of 200 nm in 25 mM phosphate buffer at pH 2.5. The analysis was performed at 25 °C and 10 kV. The developed method was validated by assessing linearity in the concentration range from 50 to 5000 ng/mL. Very good linearity was obtained, with the coefficient of determination (R2) ranging from 0.9991 to 0.9999 for both analyzed derivatives. The method demonstrated baseline resolution (Rs = 1.4). The limit of quantification ranged from 34.72 ng/mL to 34.98 ng/mL. The recoveries of all derivatives ranged from 94.8% to 100%. The total analysis time was only 6 min. The developed method enables the determination of PK20 and [Ile9]PK20 derivatives both in aqueous solutions and in serum.