ИСТИНА

Methotrexate (MTX) is an antimetabolite drug which acts by inhibiting dihydrofolatereductase, disrupting purine synthesis and preventing cell division. High circulating levels of methotrexate can cause severe myelosuppression. MTX is used to treat cancer by inhibiting cellular enzymes and preventing cell replication. MTX is the only antimetabolite for which therapeutic drug monitoring is recommended, and this is usually carried out by automated immunoassay as urgent analyses may be required. Recent clinical observations made demonstrated a negative effect on the tooth enamel of the patients who received high doses of MTX in therapy. This effect may be explained by MTX presence in saliva but report has been mentioned in the literature for determination of MTX in saliva samples HPLC-MS/MS. Saliva samples from anonymous donors were collected in prescreened cups. To 1.4 ml of saliva, 0.1 ml of the internal standard was added and pipetted into the 2-ml screw-capculture tubes, and some drops of ammonia were added to pH 9. The tubes were capped and vortexed. Initial SPE cleanup was performed on 200-mg, 3-ml Strata Screen-C cartridges (Phenomenex, USA), each conditioned with 5 ml of acetonitrile and water. The cartridges were eluted successively with 1-ml supernatant at 1 ml/min. Each extract was transferred to LC autosampler vials for injection into the LC-positive-ESI-MS/MS instrument. Liquid chromatography system (Dionex Ultimate 3000) was interfaced to an electrosryay ionization AB SciexQtrap 3200 (Canada) instrument operating in the positive ion mode. A sensitive and simple method for the methotrexate quantification in human saliva was developed using aminopterin as internal standard. An alternative sample preparation procedure allows one to achieve exceptional sensitivity, robustness and selectivity through compound quantification 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 from 3 males and 3 females with acute lymphoblastic leukemia in a prospective study, which was approved by the Moscow State University of Medicine and Dentistry. All participants, age between 40 and 58 years old, received MTX at 1 mg/kg/dose intravenously. Saliva samples were collected1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 14 h, 18 h, 24 h and 30 h after an intra-venous administration of MTX using special medical equipment and stored at −30°C. Developed novel LC–MS/MS method allows measuring MTX excretion in saliva down to a LOD of 1 ng/ml.