Savant sc250exp

Fetal plasma cortisol levels were measured using triple quadrupole mass spectrometry [17 (link)]. A total of 100 μL of internal standard (20 ng/mL cortisol-d4 in water) was added to 200 μL plasma. Steroids were extracted using 1 mL of ethyl acetate (Merck KGaA, Darnstadt, Germany). After removal of the organic supernatant, samples were dried by vacuum concentration (Savant SC250EXP, Thermo Scientific, Asheville, North Carolina, United States), re-suspended in 60 μL of mobile phase 72% methanol (Merck KGaA) and 28% water, and transferred to HPLC injector vials. A total of 12 μL was injected onto an HPLC mass spectrometer system consisting of an Accela MS pump and autosampler followed by an Ion Max APCI source on a Finnigan TSQ Quantum Ultra AM triple quadrupole mass spectrometer, all controlled by Finnigan Xcaliber software (Thermo Electron Corporation, San Jose, California, United States). The mobile phase was isocratic, flowing at 250 μL/min through a Luna HST 2.6 μm C18 (2) 100 × 3.0 mm column at 40°C (Phenomenex, Auckland, New Zealand). Retention time was 3.1 minutes for both cortisol and cortisol-d4. Ionization was in positive mode and Q2 had 1.2 mTorr of argon. The mass transitions followed were: cortisol-d4 367.2 – 121.2 at 28 V and cortisol 363.2 – 122.2 at 28 V. Mean inter- and intra-assay coefficient of variation values for cortisol were 5.8 and 6.0% respectively.

Soluble sugars (glucose, fructose, sucrose, and raffinose) were extracted four times from 20 mg (dry mass) of pulverized samples with 1.5 mL of 80% ethanol at 80°C for 20 min. The Alcohol Insoluble Residue (AIR) was dried at 45°C overnight. The supernatant was recovered, vacuum concentrated (ThermoScientific® Savant SC 250 EXP) and resuspended in 1 mL of water and 1 mL of chloroform. The soluble sugars (sucrose, fructose, glucose, and raffinose) were analyzed by High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) in a Dionex® system (ICS 5000) using a CarboPac PA1 column and eluted with 150 μM sodium hydroxide in an isocratic run of 27 min (Supplementary Figures 1, 2).

Fecal samples were collected from 41 individuals (20 very preterm and 21 terms). The samples were weighed (~100 mg) and lyophilized on a speed vac (Thermo Fisher Scientific Savant SC250EXP) overnight (24 h, 0.8 Hpa, with a −104°C refrigerated temperature trap). The wet and dry weight was noted for biomass normalization. Samples and quality controls (QC) were prepared by a tungstate precipitation. Briefly, 1,200 μL of 0.04 M Sulfuric acid (containing 15 μM L-Nor-Valine) was aliquoted into each tube containing dried sample. The mixture was vortexed (30 sec), sonicated (10 min), with repeating 2–3 times until there was a homogeneous mixture. Ceramic beads were added to samples that did not homogenize and vortexing and sonication treatment repeated. Sodium tungstate (150 μL of 10% w/v solution) was added to the homogenized samples on ice (3 min). Samples were centrifuged (20,800 g, 10 min, 4°C). The supernatant was then transferred into a 1.5 mL microfuge tube. The derivatization step was the same as for plasma samples (see above).