Background: Tobramycin is an aminoglycoside antibiotic. Due to lack of chromophores and high hydrophilicity, it is a daunting challenge to develop HPLC methods for quantification of tobramycin. Objective: To develop an LC-MS/MS method to directly determine tobramycin in M9 medium with a targeted calibration range of 50-25,000ng/mL, to support studies on an in vitro pharmacokinetic/pharmacodynamics (PK/PD) simulator for bacterial biofilms. Methods: Optimization of the method was performed in terms of chromatographic separation and mass spectrometry detection with an API 5000. Trichloroacetic acid was added into samples to improve separation. The method was validated according to the guidelines of NIH funded AIDS Clinical Trials Group Network. Results: Trichloroacetic acid not only improved separation of tobramycin but also enhanced MS signal intensity by ~3 fold. After evaluating different columns, a short Pursuit PFP column (2.0 x 50 mm, 3 mm) was used for LC separation. A gradient elution method with 20mM ammonium formate 0.14% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B) was implemented at a flow rate of 0.4mL/min: B% (time), 3 (0-0.1min)—20 (1.5min)—80 (1.51-2.0 min)—3 (2.01-3min). ESI+ and MRM with ion pair m/z 468 --&gt; 324 for tobramycin and m/z 473 --&gt; 327 for the deuterated IS were used for quantification. The calibration curve concentration range was 50—25,000 ng/mL. Matrix effect from M9 media was not significant when compared with water. Compared to the published method, this method requires a small amount of samples (only 25 mL) and short run time (only 3 min). Conclusions: Trichloroacetic aicd can be used to improve separation and MS signal for tobramycin and possibly other aminoglycosides. A sensitive and fast LC-MS/MS method was developed and validated. The small sample volume fits for the currently supported in vitro PK/PD model and pediatric patients in the future.

Trichloroacetic acid improves MS signal and separation in determination of tobramycin in M9 bacterial medium by LC-MS/MS

253rd National Meeting (2017)

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