Development and validation of a sensitive and selective LC-MS/MS method for determination of tacrolimus in oral fluids.

Tacrolimus is a commonly used immunosuppressive agent in organ transplant recipients. Therapeutic drug monitoring (TDM) of tacrolimus is essential to adjust the dose and achieve optimal immunosuppression level. Routine TDM is practiced using whole blood samples obtained through venipuncture. However, tacrolimus concentration that is present in oral fluid (OF) can theoretically represent the free or pharmacologically active form of tacrolimus. In this study, we report the development and validation of a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of tacrolimus in OF. Chromatographic separation were achieved on an Acquity UPLC BEH C18 column by a gradient elution using 2mM ammonium acetate/0.1% (v/v) formic acid in water (mobile phase A) and in methanol (mobile phase B) with a 2.2min chromatographic run time. Tacrolimus was extracted from OF with acetonitrile as the precipitating solvent. Both extraction and chromatography was optimized to provide optimal sample cleanness, negligible matrix effect, and optimal specificity. The lower limit of quantification (LLOQ) for the assay was set at 10pg/mL using a 50μL aliquot of OF obtained by passive drool. The method demonstrated adequate accuracy and precision with accuracy between 94.5-103.6%, and coefficient of variation ranging from 4 to 9.8%. Tacrolimus was stable in OF for up to one month at -80°C and the extracted matrix was stable up to 48h in auto-sampler at 20°C. The method showed high reproducibility as confirmed by incurred sample reanalysis test. This assay was employed in several clinical pharmacokinetic studies and could successfully measure the concentration of tacrolimus in OF.