Inertsustain amide column

The concentration of 1-MNA was determined using an LC-MS-8050 triple quadrupole LC-MS/MS (Shimadzu, Kyoto, Japan) coupled to an LC-30A system (Shimadzu). Chromatography was performed on an InertSustain amide column (ID 2.0 mm × 50 mm; GL Sciences) at 40 °C by means of step-gradient elution (flow rate, 0.4 mL/min) as follows: 0 to 2.0 min, 13% A/87% B; 2.0 to 2.5 min, 13% A/87% B to 50% A/50% B; 2.5 to 4.0 min, 50% A/50% B; 4.0 to 4.5 min, 50% A/50% B to 13% A/87% B; and 4.5 to 7.0 min, 13% A/87% B. A was water containing 0.1% formic acid and B was acetonitrile containing 0.1% formic acid. The mass numbers of the molecular and product ions for each compound were as follows: 1-MNA (137.2 → 94.2, CE − 22.0 V) and 1-MNA-d3 (Toronto Research Chemicals, North York, Canada) (140.0 → 97.2, CE −22.0 V). Lab solutions software (version 5.89, Shimadzu) was used for data manipulation. The detection limit was 10 ng/mL for each compound.

Determination of sialic acid content in R-10G-binding protein from 201B7 cells was performed as described previously [13 (link)]. R-10G-binding protein (5 µL, 8.5 ng) in 50 µL 50 mM hydrochloric acid was heated at 80 °C for 1 h. The hydrolysate was applied to centrifugal ultrafiltration membranes. The flow through (20 µL) was evaporated, and the sample was resuspended in 7.5 µL 75% acetonitrile. A 5 µL aliquot of the sample solution was subjected to HPLC using an InertSustain Amide Column (GL Sciences, Tokyo, Japan).
Amino sugars were analyzed as described previously [14 (link)]. Briefly, samples were subjected to hydrolysis in 6 N HCl at 100 °C for 2.5 h. Amino sugars released by hydrolysis were separated on a TSK gel SCX column (4.6 mm i.d. × 150 mm) and eluted with 0.35 M borate/NaOH buffer (pH 7.6) at 60 °C, with a high sensitivity achieved by a post-column reaction with 1% 2-cyanoacetamide. The oligosaccharides released from keratan sulfates upon keratanase II digestion or endo-β-galactosidase digestion were separated using a gel permeation HPLC system with fluorometric post-column detection [6 (link)].