(T1030-06-40) Detection and Quantification of Gentamicin in the Inner Ear Using Different Liquid Chromatography Methods

Purpose: Quantifying drugs in the inner ear is challenging due to its relative inaccessibility. The ototoxic drug, gentamicin, used to treat vestibular disorders like Meniere’s disease, consists of 4 major C-subtypes - C1, C1a, C2, and C2a. Simultaneous detection of these subtypes is difficult because gentamicin lacks UV chromophores. The volume of perilymph found in a mouse cochlea is ~1 μL, therefore we need a highly sensitive analytical method to detect and quantify the small amount of gentamicin present in the inner ear after its local delivery. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) can quantify gentamicin due to its high sensitivity and separation of components. Liquid chromatography employing UV or fluorescence detection can also quantify ortho-phthalaldehyde (OPA)-derivatized gentamicin. We present new LC-MS/MS and HPLC-fluorescence protocols to simultaneously detect multiple gentamicin subtypes.

Methods: Gentamicin was fractionated on an Acquity UPLC BEH C₁₈ column (Waters, 2.1 x 50 mm) using a solvent gradient of 0-100% acetonitrile in water containing nonafluoropentanoic acid (100 mM) at 0.2 mL/min. Gentamicin standards (1-50,000 ng/mL) were prepared in water, with amikacin (1 μg/mL) as an internal standard. MS/MS analysis was performed using electrospray ionization in positive mode and fragmented with stepped collision energy. Quantification parent < fragment ion for C1, C1a, and C2/C2a are 478.32 < 157.13 450.29 < 322.19 and 464.31 < 322.19, respectively. Alternatively, gentamicin was derivatized with OPA and ethanethiol and eluted from an ACE 5 C-18 column (250 x 4.6 mm), using an isocratic mobile phase of 0.02 M sodium heptanesulfonate monohydrate in methanol: water: acetic acid (80:18:2 (v:v:v)) at 1 mL/min. Fifty µL of gentamicin standards were combined with 700 µL HPLC-grade water and 250 µL of reagent (630 μL of a solution of 360 mg OPA dissolved in 25 mL methanol, 7.3 mL methanol, 2 mL 0.4 M boric acid, pH 10.4, 52 µL ethanethiol, kept at room temperature for 60 minutes). The solution containing gentamicin standards and the derivatizing reagents was kept at room temperature for 30 minutes before an aliquot (50 µL) was loaded onto the column. Gentamicin derivatives were detected at 337 nm-454 nm (excitation-emission wavelengths respectively) for fluorescence detection.

Results: Gentamicin can be detected at 1 ng/mL (LC-MS/MS instrument limit of detection) and quantified at 10 ng/mL (LC-MS/MS instrument limit of quantification). The standard curve obtained after plotting concentrations of gentamicin vs ratios of the area under the chromatogram peaks specific to gentamicin and internal standard was linear with R² > 0.99 (weighing is 1/x). Fragment ions were seen for each major C-subtypes of gentamicin and amikacin, with retention times of 4.00-4.13 min or 3.03 min for gentamicin and amikacin, respectively. OPA-derivatives of the four major subtypes of gentamicin, C1, C1a, C2, and C2a, incorporating ethanethiol, were successfully separated on a C₁₈ RP-HPLC column with retention times of 23.64 min, 26.51 min, 28.84 min, and 42.82 min respectively.

Conclusion: A new sensitive LC-MS/MS protocol and an HPLC-fluorescence method were developed to detect and quantify low levels of gentamicin C-subtypes in vitro. An extraction protocol will be developed, the method’s limit of detection and limit of quantification will be determined, and the method will be validated according to the USP guidelines before measuring gentamicin levels in mouse inner ear samples.

Acknowledgements:Supported by a Bellucci PreDoctoral Research Award (SD); ONR N00014-18-1-2507 subaward, and P20GM139762 (PSS).