Determination of Tobramycin in M 9 Medium by LC-MS/MS: Signal Enhancement by Trichloroacetic Acid.

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 M-9 media was developed. Aliquots of 25 mu L M-9 media samples were mixed with the internal standard (IS) tobramycin-d(5) (5 mu g/mL, 25 mu L) and 200 mu L 2.5% trichloroacetic acid. The mixture (5 mu L) was directly injected onto a PFP column (2.0 x 50 mm, 3 mu m) 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 M 9 media was not significant when compared with injection solvents, but signal enhancement by trichloroacetic acid was significant (similar to 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.