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

Vet World Vol.19 January-2026 Article - 2

Research Article

Veterinary World, 19(1): 15-28

https://doi.org/10.14202/vetworld.2026.15-28

Anisa Bardhi¹, Domenico Ventrella^1,2, Alberto Elmi^1,3, Ronette Gehring⁴, Davide Martelli⁵, Ilaria Troisio⁶, Maria Laura Bacci^1,2, and Andrea Barbarossa^1,2

1. Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (BO), Italy.

2. Health Sciences and Technologies-Interdepartmental Centre for Industrial Research (CIRI-SDV) - University of Bologna, Ozzano dell’Emilia (BO), Italy. .

3. Department of Veterinary Sciences, University of Pisa, Pisa, Italy. .

4. Department of Population Health Sciences, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands. .

5. Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy. .

6. Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (BO), Italy. .

Background and Aim: 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-hydroxypropranolol, 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.

Materials and Methods: 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).

Results: 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.

Conclusion: 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.

Keywords: 4-hydroxypropranolol, beta-blocker, dried blood spots, liquid chromatography–tandem mass spectrometry, microsampling, Pharmacokinetics, pigs, Propranolol.

How to cite this article: Bardhi A, Ventrella D, Elmi A, Gehring R, Martelli D, Troisio I, Bacci ML, Barbarossa A. 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. Vet World. 2026;19(1): 15–28.

Received: 23-07-2025 Accepted: 03-12-2025 Published online: 06-01-2026

Corresponding author: Andrea Barbarossa E-mail: andrea.barbarossa@unibo.it

DOI: 10.14202/vetworld.2026.15-28

Copyright: Bardhi, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.