6470 series triple quadrupole mass spectrometer

Metabolites from powderized flash-frozen brain tissue were extracted in ice-cold 80% methanol. Subsequently, extract from the equivalent of 1 mg tissue was dried down in a room temperature speed-vac for metabolomics analysis. Metabolites were resolved using reverse phase ion-paired chromatography on an Agilent 1290 Infinity II Series LC and detected by MRM in negative ion mode on an Agilent 6470 series triple quadrupole mass spectrometer. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) targeted MRM method used for metabolomics analysis was developed and implemented by Agilent Technologies.

The hMAO-A and hMAO-B enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reaction mixture contained hMAO-A (2.5 µg/mL protein final concentration) or hMAO-B (6.25 µg/mL protein final concentration) enzyme and tested compound in final concentration of 1 and 10 µM in 50 mM potassium phosphate buffer with 20% (v/v) glycerol (pH 7.5). The mixture was pre-incubated at 37 °C for 5 min and subsequently substrate kynuramine was added to the final concentration of 60 µM in the case of hMAO-A and 30 µM in the case of hMAO-B. The final volume of reaction mixture was 0.1 mL. The whole reaction mixture was incubated at 37 °C for 30 min. The reaction was stopped by the addition of 200 µL acetonitrile/methanol mixture (ratio 1:1) and cooling down to 0 °C. The sample was then centrifuged (16.500× g) for 10 min. The deamination product of kynuramine formed during the enzymatic reaction 4-hydroxyquinoline (4-HQ) was determined by HPLC–MS on a 2.1 mm × 50 mm, 1.8 µm Zorbax RRHD Eclipse plus C18 column (Agilent) by using a 6470 Series Triple Quadrupole mass spectrometer (Agilent) (electrospray ionisation – positive ion mode). Three MRM transitions were followed for kynuramine (165.1 = > 30.2, 165.1 = > 118.0, 165.1 = > 136.0) and 4-HQ (146.1 = > 51.1, 146.1 = > 77.0, 146.1 = > 91.0). Eluents: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile.

An aliquot of fresh leaves (300 mg) was homogenized in 80% ethanol and heated at 80 °C for 20 min. The homogenate was centrifuged at 4500 rpm for 10 min. The supernatant was collected in a clean tube, the pellet was resuspended in 50% ethanol and reheated at 80 °C for 20 min. After centrifugation, the supernatants were pooled. This step was repeated once more. The content of sugars (glucose, fructose, sucrose) and sugar alcohols (sorbitol, glycerol) in the combined supernatants determined using UHPLC Infinity II 1290 system (Agilent Technologies, Santa Clara, CA, USA) using a 6470 Series Triple Quadrupole mass spectrometer (Agilent Technologies; electrospray ionization, negative polarity) as detector. The method used a BEH Amide column (2.1 mm × 150 mm, 2.6 µm, Waters, Milford, MA, USA). An isocratic elution program was used for chromatographic separation applying mobile phase comprising 20:80% mixture of 0.05% formic acid in water and 8:2 acetonitrile/methanol mixture. The flow rate was 0.4 mL min⁻¹. Ion source parameters: gas temperature 150 °C, gas flow 6 L min⁻¹, nebulizer 40 psi, sheath gas temperature 300 °C, sheath gas flow 128 L min⁻¹, capillary voltage 2500 V, and nozzle voltage 0 V. The sample injection volume was 1 µL. All standards used were purchased from Sigma Aldrich (Darmstadt, Germany).