Pure myo inositol

The determination of free deuterated and natural inositol in the medium was performed by RP–PGC–TOFMS, using known amounts of pure myo-inositol (Sigma-Aldrich) as standards. The medium samples were prepared by centrifugal filtration (60 min, 10 000 rpm, 4 °C; Amicon Ultra, 3 kDa MW cut-off) and diluted 1:100 in LC–MS grade H₂O (Sigma Aldrich) prior to injection. A detailed description of the chromatographic separation is provided elsewhere [29 (link)]. The chromatography column was coupled on-line to a time-of-flight mass spectrometer (Agilent 6230 LC–TOFMS) via a Dual AJS ESI spray chamber. ESI was performed in negative mode (45 psig nebulizer pressure, 350 °C sheath gas temperature, 11 L/min sheath gas flow, 250 °C drying gas temperature, 12 L/min drying gas flow, 110 V fragmentor voltage, 60 V skimmer voltage, 3500 V capillary voltage). MS data was acquired in the mass range 50-1000 m/z at an acquisition rate of 3 spectra per second. Both unlabeled and D₆-labeled myo-inositol were detected as formic acid adducts ([M + COO⁻]⁻, 225.0616 m/z and 231.0993 m/z, respectively) at a retention time of 3.9 min. After peak integration (Agilent MassHunter Quantitative Analysis 7.0, extraction window ±5 ppm), the peak areas were used to calculate the ratio between D₆-labeled and unlabeled inositol.