Beh amide uhplc column

The plasma, urine, and HM samples were stored at less than −20°C and shipped frozen for analysis to Metabolon, Inc (Durham, NC). No more than 1 sample per subject per time point was analyzed for plasma, urine, and HM. HM samples were first diluted 1:500 in water. All of the samples were spiked with an internal standard and subjected to protein precipitation with methanol. Following centrifugation, supernatant was removed. Plasma supernatant was further evaporated to dryness and reconstituted in methanol:water (75:25, vol/vol). Aliquots of urine and HM supernatant, reconstituted plasma extract, and freshly prepared calibration standards were injected onto an Agilent 1290/AB Sciex QTrap 5500 liquid chromatography with tandem mass spectrometry system (AB Sciex, Framingham, MA) equipped with a BEH Amide UHPLC column (Waters Corporation, Milford, MA). Data were acquired using electrospray ionization in negative ionization mode. 2′FL concentrations were calculated based on the area ratios of 2′FL and internal standard peaks using a weighted (1/×) least squares regression analysis generated from external calibration standards included in each run.

Functional
groups and concentrations of specific compounds present within the
supernatant were characterized via a combination of pH testing with
a handheld pH meter, FTIR, and liquid chromatography–mass spectroscopy
(LC–MS).
A drop of the filtered sample supernatant was
placed on a PerkinElmer spotlight 400 FTIR attenuated total reflectance
(ATR) crystal, and absorption was measured between 4000 and 650 cm^–1. To better display absorption characteristics of
the mixed products, DI water was measured, and its spectrum was subtracted
from those of the supernatants.
LC–MS was performed on
a Q-Exactive HF mass spectrometer
with a U3000 chromatography system (Thermo Fisher Scientific); samples
were prepared at 20 μg/mL in 1:1 acetonitrile/water and run
for 30 min at 35 °C through a Waters BEH Amide UHPLC column (2.1
× 100 mm). Mobile phase A was composed of 80:20 MeCN/H₂O with 0.1% NH₄OH, and mobile phase B was identical except
for a 70:30 ratio of MeCN/H₂O. Commercially available standards
for glucose, fructose, galactose, mannose, xylose, ribose, arabinose,
deoxyglucose, deoxyribose, and glutaric acid were analyzed as external
standards, and a ¹³C-labeled glucose compound was run across
all samples as an internal standard.