Sensitive Isocratic RP-HPLC–Fluorescence Method for OPA-Ethanethiol Derivatized Gentamicin in Small-Volume Biological Samples

Research conducted by the Bellucci Translational Hearing Center, part of the Department of Biomedical Sciences at Creighton University School of Medicine (Omaha, Nebraska) yielded a new, simple, fast, and sensitive isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the detection and quantification of fluorescent o-phthalaldehyde (OPA)-ethanethiol derivatized gentamicin (a broad-spectrum aminoglycoside used frequently to treat gram-positive and gram-negative bacterial infections) in very small biological sample volumes. To the research team’s knowledge, there is no previous report regarding the use of ethanethiol for the derivatization of gentamicin with OPA, and the simultaneous determination of the four major C-subtypes of gentamicin using OPA-derivatives. A paper based on their work was published in the Journal of Pharmaceutical and Biomedical Analysis.¹

An idiopathic inner ear disorder, the symptoms of Ménière’s disease include episodic vertigo attacks, fluctuating low-frequency hearing loss, aural fullness, and tinnitus.² While previous research has determined that gentamicin injections produces variable degrees of vestibular dysfunction, reducing the sensitivity of peripheral vestibular sensory cells and, importantly, alleviating the clinical symptoms of Ménière’s disease, dose-dependent drug-induced damage within the inner ear cannot be accurately predicted due to the inner ear being embedded deep inside the temporal bone.^3-5 As gentamicin does not have UV-absorbing chromophores or fluorophores, thus preventing the use of traditional RP-HPLC with ultra-violet-visible light (UV–vis) or fluorescence (Fl) detection often found in conventional quality-controlled clinical pathology laboratories, the need for a new method was clear to the Creighton researchers.^6-8

The research team achieved optimum chromatographic conditions for their study on a C₁₈ column with a mobile phase consisting of methanol, glacial acetic acid, and an aqueous solution of sodium 1-heptanesulfonate at a flow rate of 1.0 mL/min under ambient conditions. The team reports that the method was successfully validated according to the acceptance criteria of USP guidelines in terms of selectivity, linearity, accuracy, precision, and sensitivity. The linearity of the method was demonstrated with a concentration range of gentamicin (10-400 ng/mL) prepared in artificial perilymph. The limit of detection was 0.2 ng/mL and the limit of quantification was 10-11 ng/mL for all four major C-subtypes of gentamicin. Finally, due to its high sensitivity, this method was successfully applied to quantify gentamicin concentrations in the small volumes of perilymph present in the inner ear of mice.¹

“Thus,” the authors of the article write,¹ “this RP-HPLC-fluorescence method for detecting derivatized gentamicin in preclinical models is promising in terms of simplicity and high sensitivity.”

While the researchers state that their method assay is effective for detecting and quantifying gentamicin in small volumes of biological fluids with low protein content, there are some limitations which should be considered. For example, the low extraction efficiency of gentamicin from tissue samples and high sample protein content may interfere with the analysis and compromise sensitivity. In addition, the need to derivatize samples adds complexity and time to the protocol. The authors of the study state that these limitations highlight the need for careful sample preparation and consideration of matrix effects when applying this method to more complex biological samples.¹

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References

1. Dash, S.; McDevitt, M. T.; Smith, D. D. et al. Determination of Gentamicin C-Subtypes in Inner Ear Perilymph Using Liquid Chromatography with Fluorescence Detection. J Pharm Biomed Anal. 2026, 273, 117394. DOI: 10.1016/j.jpba.2026.117394
2. Mohseni-Dargah, M.; Falahati, Z.; Pastras, C. et al. Meniere's Disease: Pathogenesis, Treatments, and Emerging Approaches for an Idiopathic Bioenvironmental Disorder. Environ. Res. 2023, 238 (Pt1), 116972. DOI: 10.1016/j.envres.2023.116972
3. Webster, K. E.; Galbraith, K.; Lee, A. et al. Intratympanic Gentamicin for Meniere's Disease. Cochrane Database Syst. Rev. (Online) 2023, 2 (2), CD015246, DOI:10.1002/14651858.CD015246.pub2
4. Cohen-Kerem, R.; Kisilevsky, V.; Einarson, T. R. et al. Intratympanic Gentamicin for Meniere's Disease: A Meta-Analysis. Laryngoscope 2004, 114 (12), 2085-2091. DOI: 10.1097/01.mlg.0000149439.43478.24
5. H. Li, H.; S. Agrawal, S.; S.A. Rohani, S. A. et al. Unlocking the Human Inner Ear for Therapeutic Intervention. Sci. Rep. 2022, 12 (1), 18508. DOI: 10.1038/s41598-022-22203-2
6. Joseph, A. Rustum, A. Development and Validation of a RP-HPLC Method for the Determination of Gentamicin Sulfate and its Related Substances in a Pharmaceutical Cream Using a Short Pentafluorophenyl Column and a Charged Aerosol Detector. J. Pharm. Biomed. Anal. 2010, 51 (3), 521-531. DOI: 10.1016/j.jpba.2009.09.002
7. Farouk, F.; Azzazy, H. M.; Niessen, W. M. Challenges in the Determination of Aminoglycoside Antibiotics, A Review. Anal. Chim. Acta 2015, 890, 21-43. DOI: 10.1016/j.aca.2015.06.038
8. E. Herrero-Hernandez, E.; D. Garcia-Gomez, D.; I. Ramirez Perez, I. et al. Determination of Aminoglycosides by Ion-Pair Liquid Chromatography with UV Detection: Application to Pharmaceutical Formulations and Human Serum Samples. Molecules 2024, 29 (13). DOI: 10.3390/molecules29133210