PhD Scientific Days 2026

Poster Session 2.G - Pharmaceutical Sciences and Health Technologies

Quantification of Gentamicin in Rat Plasma and Urine Using LC-MS/MS: Method Development and Validation

Name of the presenter

Széles, Aliz

Institute/workplace of the presenter

Doctoral College Semmelweis University; HUN-REN Research Centre for Natural Sciences; Toxi-Coop Toxicological Research Centre

Authors

Aliz Széles¹, Katalin Monostory², Tibor Renkecz³
1: Doctoral College Semmelweis University; HUN-REN Research Centre for Natural Sciences; Toxi-Coop Toxicological Research Centre
2: HUN-REN Research Centre for Natural Sciences, Magyar Tudósok körútja 2., H-1117 Budapest, Hungary
3: Toxi-Coop Toxicological Research Centre, Magyar jakobinusok tere 4., H-1122 Budapest, Hungary

Text of the abstract

Gentamicin (GEN) is a broad-spectrum aminoglycoside antibiotic with key applications in the treatment of severe bacterial infections, including sepsis and endocarditis. Its bactericidal activity against aerobic Gram-negative pathogens makes it an important agent in antimicrobial therapy. Accurate quantification of GEN in biological matrices is essential for therapeutic drug monitoring to optimize efficacy and reduce its nephrotoxic and ototoxic side effects.
A reverse-phase high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of GEN in rat plasma and urine, using kanamycin as the internal standard (IS), with a calibration range of 20-2000 ng/mL. Method validation was performed according to the ICH M10 bioanalytical guideline. Gradient elution was performed using mobile phase A (5% ammonium solution in ultrapure water) and mobile phase B (acetonitrile) on a SupelTM Carbon LC column (10 cm x 2.1 mm, 2.7 μm) with a guard column (2 cm x 2.1 mm, 2.7 μm). A Shimadzu LCMS-8060 triple quadrupole mass spectrometer was operated in multiple reaction monitoring mode with an electrospray ionization source, selecting the monoprotonated precursor ion for fragmentation.
Plasma and urine samples were stable at - 75 °C ±10 °C for at least 83 and 8 days, respectively, as well as across three freeze-thaw cycles, and 4 hours at room temperature. Mean recoveries were 114-89.5%, 109-106%, 111-91.9%, and 110-102% at high, medium, low and IS concentration levels, respectively.
Chromatograms demonstrated clear separation of GEN and the internal standard. Accuracy and precision remained within ±15% across all concentrations. No significant matrix effect or carryover were detected.
The developed and validated LC-MS/MS method enables sensitive and selective quantification of GEN in rat plasma and urine. This technique can support both pharmacokinetic studies and research into drug-induced toxicity or dosage optimization, offering insights into the clinical efficacy and safety profile of GEN.
This work was supported by the 2024-2.1.2-EKÖP-KDP-2024-00002 University Research Scholarship Programme of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund. Additional funding was secured through the Semmelweis 250+ Excellence PhD Scholarship.