Quantification of Formate by GC-MS

Formate quantification using gas chromatography-mass spectrometry (GC-MS) was performed as previously described by ref. ³⁴ . Media samples from CML CD34⁺ cells (40 μL) were added in 20 μl of 50 μM internal standard sodium ¹³C,²H-formate (m + 2), 10 μl of 1 M sodium hydroxide, 5 μl of benzyl alcohol and 50 μl of pyridine. While vortexing, derivatisation of formate was commenced by the addition of 20 μl of methyl chloroformate. 200 μl of water and 100 μl of methyl tertiary butyl ether were then added, and the samples were vortexed for 20 s and centrifuged at 10,000 g for 5 min. The resulting apolar phase containing the formate derivative (benzyl formate) was transferred to a GC-vial and capped. Formate standards and blank samples (water) were prepared in the same manner and analysed with the experimental samples. Derivatized formate samples were analysed with an Agilent 7890B GC system coupled to a 7000 triple quadrupole MS system³⁴ . MassHunter Quantitative analysis software (Agilent Technologies) was used to extract and process the peak areas for m + 0, m + 1, m + 2 formate. After correction for background signals, quantification was performed by comparing the peak area of formate (m/z of 136) and of m + 1 formate (m/z of 137) against that of m + 2-formate (m/z of 138).