Abstract
Two new and highly sensitive liquid chromatography techniques were developed to detect and quantify the four major C-subtypes of gentamicin in microliter samples of artificial perilymph or perilymph collected from mice. The new ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method and high-performance liquid chromatography with fluorescence detection (HPLC-Fl) assays allowed the simultaneous detection and quantification of gentamicin subtypes, with the HPLC-Fl showing superior sensitivity, speed, and suitability for perilymph samples. The HPLC-Fl assay was validated according to USP guidelines and demonstrated excellent linearity, specificity, precision, and accuracy for detecting gentamicin in the perilymph, with a detection limit of ≤0.2 ng/mL for the major C-subtypes of gentamicin. Thus, it is a highly efficient tool for measuring drug levels in small biological volumes. The HPLC-Fl technique was used to quantify gentamicin C-subtypes concentrations in murine perilymph and serum following local (intratympanic) and systemic (intraperitoneal) administration. The data obtained was used to determine the pharmacokinetics of gentamicin in perilymph and obtained crucial insights into how gentamicin is absorbed and distributed in the perilymphatic space before clearance from this space in the murine inner ear. The pharmacokinetic profile of gentamicin in murine perilymph revealed that its concentration peaks within an hour after intratympanic administration and then declines rapidly. Gentamicin C1 exhibited the fastest clearance from perilymph, while C2 (most ototoxic subtype) or C2a was retained for a longer duration in the perilymph. In contrast, after intraperitoneal administration, gentamicin concentrations in perilymph peaked more slowly, with a half-life of approximately four hours and complete elimination from the perilymph within 13 hours. The most ototoxic subtype, C2, had the highest concentration and a longer mean residence time compared to C1.
Following intratympanic administration, the level of gentamicin in the serum was extremely low. The most ototoxic subtype C2, had the highest level in the serum, and C1 had the lowest. After intraperitoneal administration, gentamicin levels peaked immediately in the serum, followed by a rapid decline. C2 maintained the highest concentration in the serum, posing a potential risk of ototoxicity and nephrotoxicity.
These findings have therapeutic implications for treating inner ear disorders, such as Ménière’s disease. Intratympanic administration allows for higher drug concentrations directly at the site of action, offering potential benefits for targeting the vestibular hair cells, particularly for vertigo management. However, at higher concentrations, this delivery method increases the risk of cochleotoxicity. This study is significant as it highlights the distinct pharmacokinetic profiles of each C-subtype, which is critical for optimizing drug delivery strategies for inner ear disorders, offering insights into how to achieve therapeutic efficacy while minimizing the risk of ototoxicity.