Development and validation of LC-MS/MS methods to measure tobramycin and lincomycin in plasma, microdialysis fluid and urine: application to a pilot pharmacokinetic research study

Background: The aim of our work was to develop and validate a hydrophilic interaction liquid chromatography- electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) methods for the quantification of tobramycin (TMC) and lincomycin (LMC)in plasma, microdialysis fluid and urine. Methods: Protein precipitation was used to extract TMC and LMC from plasma, while microdialysis fluid and urine sample were diluted prior to instrumental analysis. Mobile phase A consisted of 2 mM ammonium acetate in 10% acetonitrile with 0.2% formic acid (v/v) and mobile phase B consisted of 2 mM ammonium acetate in 90% acetonitrile with 0.2% formic acid (v/v). Gradient separation (80%-10% of mobile phase B) for TMC was done using a SeQuant zic-HILIC analytical guard column. While separation of LMC was performed using gradient elution (100%-40% of mobile phase B) on a SeQuant zic-HILIC analytical column equipped with a SeQuant zic-HILIC guard column. Vancomycin (VCM) was used as an internal standard. A quadratic calibration was obtained over the concentration range for plasma of 0.1-20 mg/L for TMC and 0.05-20 mg/L for LMC, for microdialysis fluid of -0.1-20 mg/L for both TMC and LMC, and 1-100 mg/L for urine for both TMC and LMC. Results: For TMS and LMC, validation testing for matrix effects, precision and accuracy, specificity and stability were all within acceptance criteria of +/- 15%. Conclusions: The methods described here meet validation acceptance criteria and were suitable for application in a pilot pharmacokinetic research study performed in a sheep model.