# First validated liquid chromatography–tandem mass spectrometry method for simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots and its application to a pharmacokinetic study

## TL;DR

This study developed the first validated method to measure propranolol and its metabolite in pigs, revealing differences in drug metabolism compared to humans.

## Contribution

First validated LC–MS/MS method for quantifying propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots.

## Key findings

- The method showed excellent linearity and accuracy for both compounds in the tested concentration ranges.
- 4-hydroxypropranolol was undetectable in pig plasma, indicating species-specific metabolic differences.
- The developed method is suitable for pharmacokinetic studies in pigs due to its simplicity and compatibility with microsampling.

## Abstract

Propranolol is a widely used non-selective beta-adrenergic blocker in human medicine, with well-characterized pharmacokinetics (PK) in humans but virtually no data available for pigs, a species of growing biomedical relevance. Furthermore, no validated bioanalytical methods exist for propranolol or its primary metabolite, 4-hydroxy-propranolol, in porcine matrices. This study aimed to develop and validate a rapid, sensitive, and reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of propranolol and 4-hydroxypropranolol in pig plasma and dried blood spots (DBS), and to apply it in a preliminary PK investigation in pigs.

Sample preparation involved simple protein precipitation (plasma) or solvent extraction (DBS) using acetonitrile–water mixtures, followed by chromatographic separation on a Bridged ethyl hybrid C18 column (50 × 2.1 mm, 1.7 μm; 4-min run). Detection was performed in Multiple reaction monitoring mode with propranolol-d7 as the internal standard. Validation followed EMA ICH M10 guidelines, assessing linearity, accuracy, precision, matrix effects, recovery, and stability. The method was then applied to plasma samples from five juvenile female pigs receiving oral propranolol (3 mg/kg, q8 h).

The method demonstrated excellent linearity (r2 > 0.99) and acceptable accuracy and precision (±15%) across 2–500 ng/mL (propranolol) and 1–400 ng/mL (4-hydroxypropranolol). Recoveries ranged from 83% to 116% (plasma) and 81%–113% (DBS), with no matrix interference or carry-over. In vivo PK data revealed rapid absorption (Tmax 1.14 ± 0.63 h), moderate elimination (t½ 2.19 ± 0.86 h), and a mean Cmax of 112.02 ± 81.87 ng/mL. Notably, 4-hydroxypropranolol was undetectable in all plasma samples, suggesting species-specific metabolic differences.

This study reports the first validated LC–MS/MS assay for propranolol and 4-hydroxypropranolol in pigs and demonstrates its successful application in a PK study. The method’s simplicity, short runtime, and compatibility with DBS microsampling make it ideal for preclinical and veterinary research, minimizing animal stress and sampling volume. Absence of 4-hydroxypropranolol highlights interspecies metabolic variability and warrants further investigation into propranolol biotransformation pathways in swine and other translational models.