Targeted Metabolite Quantification by Mass Spectrometry

Metabolites nicotinamide, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), AMP and ATP were measured using tandem mass spectrometry^{30 (link),39 (link)}. Metabolite extracts using 80% methanol (−80 °C) were dried by nitrogen. Samples were re-suspended using 20 μl LC/MS grade water, of which 10 μl were injected and analysed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/Sciex) coupled to a Prominence HPLC system (Shimadzu) via selected reaction monitoring (SRM). The dwell time was 4 ms per SRM transition and the total cycle time was 1.89 s. Approximately 8 to 11 data points were acquired per detected metabolite. Samples were delivered to the MS using a 4.6 mm internal diameter × 10 cm Amide XBridge HILIC column (Waters) at 300 μl min⁻¹. Gradients were run starting from 85% buffer B (HPLC grade acetonitrile) to 35% B from 0 to 3.5 min; 35% B to 2% B from 3.5 to 11.5 min; 2% B was held from 11.5 to 16.5 min; 2% B to 85% B from 16.5 to 17.5 min; 85% B was held for 7 min to re-equilibrate the column. Buffer A consisted of 20 mM ammonium hydroxide and 20 mM ammonium acetate (pH 9.0) in 95:5 water:acetonitrile. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v2.0 software (AB/Sciex).