Raptor arc 18 column

Freeze dried fruit powder was analyzed for vitamin C using a Thermo Vanquish UHPLC-PDA system (Thermo Fisher Scientific, Waltham, MA, USA) with a Waters^® Acquity HSS T3 column (150 × 2.1 mm, 1.8 μm) (Waters, Rydalmere, NSW, Australia) according to the method reported by Phan et al. [125 (link)], with slight modifications. Briefly, 100 mg fruit powder was extracted with 2 mL solvent containing 8% acetic acid, 3% metaphosphoric acid and 1 mM ethylenediaminetetraacetic acid tetrasodium salt three times under vortexing, sonication, shaking and centrifugation. Supernatants were combined and filtered using a 0.22 um GHP membrane filter (Pall, Melbourne, VIC, Australia) before analysis. Dehydroascorbic acid (DHAA) in the sample was reduced using DL-Dithiothreitol. Total vitamin C (L-AA + DHAA) was determined at 245 nm and 25 °C with isocratic elution (aqueous 0.1% formic acid at 0.2 mL/min).
Folate vitamers were analyzed following a stable isotope dilution assay according to the method described by Striegel, Chebib, Netzel and Rychlik [61 (link)], using a UHPLC-MS/MS (Shimadzu, Rydalmere, NSW, Australia), equipped with a Raptor ARC-18 column (Restek, Bellefonte, PA, USA). The folate derivatives measured included pteroylmonoglutamic acid (PteGlu), 5-methyltetrahydrofolate (5mTHF), 5-formyltetrahydrofolate (5fTHF), 10-formyl-pteroylglutamic acid (10f PteGlu) and tetrahydrofolate (THF).

Reactions were performed in a 100-μl final volume of Arr buffer (20 mM Tris-HCl [pH 7.9], 40 mM KCl, and 0.5 mM MgCl₂). M. smegmatis or M. abscessus Arr (10 μM) was mixed with Rif or KglA (100 μM) in 80 μl of Arr buffer at 37°C for 10 min. A 20-μl aliquot of NAD⁺ in Arr buffer was added (250 μM final) and incubated for 1 h at 37°C. The reaction was quenched with 500 μl of methanol (MeOH). Methanol was then evaporated under negative pressure and the reaction mixture analyzed by LC-MS. All analytical separations were performed on an Agilent 1260 HPLC instrument by injection of 1 to 5 μl of sample onto a Raptor ARC-18 column (150 mm by 2.1 mm) (Restek) or an Ultra C₄ column (150 mm by 2.1 mm) operated at 0.2 μl/min and then eluted using a 30-min linear gradient from 5% to 100% of acetonitrile. The mobile phase was supplemented with 0.1% formic acid. Mass spectra were recorded in positive-ion mode on a Bruker MicrOTOF II time-of-flight mass spectrometer.