A validated LC–MS/MS method for rapid determination of methotrexate in human saliva and its application to an excretion evaluation study

• We study the determination of methotrexate in saliva samples by LC–MS/MS.
• The developed assay was based on the use of solid-phase extraction.
• The method has a detection limit (LOD) of 1 ng/ml.
• Application of this procedure was demonstrated in the saliva excretion study.

A sensitive and simple method for the methotrexate quantification was developed using aminopterin as internal standard. Methotrexate is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma. The compound was quantified by liquid-chromatography coupled to electrospray ionization (positive ion-mode) low-energy collision dissociation-tandem mass spectrometry. Quantitative detection was by multiple reaction monitoring of the transitions of the [M + H]+ ion of MTX to its common product ion at m/z 308.4 and of aminopterin at m/z 441.2 → m/z 294.0. The method demonstrated linearity over at least three orders of magnitude and had a detection limit of 1 ng/ml for methotrexate. A run time of less than 8.0 min for each sample made it possible to analyze a large number of human saliva samples per day. Application of this procedure was demonstrated to a saliva excretion study of methotrexate on the samples obtained after an intravenously administration of 1 mg/kg/dose of methotrexate to six patients with acute lymphoblastic leukemia