Hpae pad ics 5000

Quantification of eight neutral (LNDFH, LDFT, 2′-FL, LNFP-I, LNT, LNnT, N-acetylgalactosaminyllactose, and 3-Hex) and 3 acidic (6′-SLN, 6′-SL, and 3′-SL) OSs was carried out using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD ICS-5000; Thermo Fisher Scientific, Waltham, MA, USA). Diluted OS solutions were filtered through a 0.22 μm membrane and injected using a 25 μL loop. Chromatographic separation was carried out on a CarboPac PA200 analytical column (3 mm × 250 mm; Dionex, Sunnyvale, CA, USA) and a CarboPac PA200 guard column (3 mm × 50 mm; Dionex, Sunnyvale, CA, USA) with 0.5 mL/min elution and a non-isocratic gradient: 0–10 min 50% B, 10–50 min 45% B – 10% C. The column was equilibrated for 5 min with 10% B followed by 10 min with 50% B. Solvent A was deionized water, solvent B 200 mM NaOH, and solvent C was 100 mM NaAc in 100 mM NaOH. Quantification was assessed by external calibration using a mixture of all OS standards ranging from 0.0001 to 0.03 g/L.

Bacterial cultures in mMRS medium were collected at 6 and 30 h of growth, representing the early-log phase and the late stationary phase. Bacterial cells were removed by centrifugation at 10,000× g for 2 min. Supernatant was collected and diluted 10,000-fold. Dilutions were filtered through a 0.22 μm cellulose acetate membrane (VWR International, Radnor, PA, USA) and 25 µL of these supernatants were injected into a High-Performance Anion-Exchange Chromatograph Coupled with Pulsed Amperometric Detection instrument (HPAE-PAD ICS-5000, Thermo Scientific, Sunnyvale, CA, USA) according to methods from [43 (link)] with some modifications. Briefly, chromatographic separation was carried out on a CarboPac PA1 analytical column (4 × 250 mm, DionexTM, ThermoFisher Scientific, Waltham, MA, USA) and CarboPac PA1 guard column (4 × 50 mm, Dionex) with an isocratic gradient, 0–30 min 73.5% A, 25% B, 1.5% C, at a 1.0 mL/min flow rate, where solvent A was deionized water, solvent B 100 mM NaOH and solvent C was 500 mM NaOAc in 100 mM NaOH. HMOs were quantified using calibration curves generated using reference standards of LNT, LNnT (Dextra, Reading, UK) and 2′-FL (Carbosynth, St. Gallen Switzerland), ranging in concentration from 0.00025 to 0.005 mg/mL. All samples were analyzed in triplicate.