Determination of Tobramycin in M9 Medium by LC-MSMS: Signal Enhancement by Trichloroacetic Acid

Abstract EN

It is well known that ion-pairing reagents cause ion suppression in LC-MS/MS methods.

Here, we report that trichloroacetic acid increases the MS signal of tobramycin.

To support studies of an in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms, an LC-MS/MS method for determination of tobramycin in M9 media was developed.

Aliquots of 25 μL M9 media samples were mixed with the internal standard (IS) tobramycin-d5 (5 µg/mL, 25 µL) and 200 µL 2.5% trichloroacetic acid.

The mixture (5 µL) was directly injected onto a PFP column (2.0 × 50 mm, 3 µm) eluted with water containing 20 mM ammonium formate and 0.14% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid in a gradient mode.

ESI+ and MRM with ion m/z 468 → 324 for tobramycin and m/z 473 → 327 for the IS were used for quantification.

The calibration curve concentration range was 50–25000 ng/mL.

Matrix effect from M9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (∼3 fold).

The method is simple, fast, and reliable.

Using the method, the in vitro PK/PD model was tested with one bolus dose of tobramycin.