Xbridge amide column

LC-MS measurements were done as described in [18 (link)] with 10 μl injection volumes. In short, an HPLC (1200 Infinity, Agilent, Santa Clara, CA, USA) equipped with XBridge Amide Column (3.5 μm particle size, 2.1 × 150 mm, Waters, Milford, MA, USA) was used. Buffers were A) 70/30 Milli-Q H₂O/Acetonitrile supplemented with 0.1% NH₄OH and B) 80/20 Acetonitrile/Milli-Q H₂O supplemented with 0.1% NH₄OH. The gradient used was 0% to 60% buffer A in 15 min, then 60% to 0% buffer A in 3 min. The sugars were detected using Q-TOF MS (6530, Agilent, Santa Clara, CA, USA) in the negative ion mode. Lactose, 2’-FL and 3-FL were used as standards in concentrations between 1 ppm to 100 ppm. This LC setup was also used to purify the sugar for NMR studies, with the following modifications: The gradient was 0% to 60% buffer A in 10 min, then 60% to 0% buffer A in 1 min and equilibration of 4 min between injections. Injections of 35 μl containing approximately 3000 ppm of the product were used and the fucosyllactose fraction (time 8 min to 10 min) was collected prior to the MS-detector.

NGLs were fractionated by normal phase HPLC with a XBridge Amide column (3.5 μm, 4.6 × 250 mm, Waters, Borehamwood, UK) using a Gilson system coupled with a fluorescence detector (λ_ex at 255 nm and λ_em at 405 nm) (22 (link)). The gradient was CHCl₃/MeOH/H₂O 130:70:9 (solvent A) to 10:20:8 (solvent B) in 60 min at a flow rate of 0.5 ml/min.

Culture samples were primarily analyzed by TLC on Silica gel 60 F₂₅₄ precoated plates (Merck, Germany). All plates were run in a closed TLC chamber and developed using standard visualization techniques and agents: UV fluorescence (254 nm) or by staining with 10 % (v/v) H₂SO₄ and subsequent charring. The mobile phase for detecting the various flavonols and corresponding glycosides consisted of an ethyl acetate:acetic acid:formic acid:water (100:11:11:27 v/v) mixture [63 (link)]. Product spot intensities of other flavonol glycosides were processed and quantified using ImageJ [64 ]. HPLC quantification of sucrose, fructose and glucose was performed using an X-bridge Amide column (35 μm, Waters, USA) as described previously [45 ]. Quercetin, hyperoside, quercitrin and isoquercitrin were detected with the method described by Pandey et al. [41 (link)] using a Varian HPLC system (Agilent technologies, California). Mass spectrometry for determination of the various flavonol glycosides was performed with a Micromass Quattro LC (McKinley Scientific, USA). Detection was performed in negative mode ESI-224 MS with a capillary voltage of 2.53 kV, a cone voltage of 20 V, cone and desolvation gas flows of 93 and 420 L/h, and source and cone temperatures of 150 and 350 °C, respectively.