Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver

Abstract

Source: Cao, C. et al. Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. J. Vis. Exp. (2017)

In this video, we demonstrate the detection of sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, using high-performance liquid chromatography, HPLC, following derivatization with a suitable fluorescent labeling reagent to aid in fluorescence detection.

Protocol

1. Fluorescence Derivatization of Sialic Acids Prepare 10 mL of OPD-solution, consisting of 100 mg of o-phenylenediamine and 208 mg of sodium hydrogen sulfite in 10 mL of distilled  H₂O. Add 20 µL of OPD-solution to the sialic acid samples. Vortex vigorously for 30 s and incubate samples for 4 h at 80 °C in the dark (i.e. wrap the microtubes in aluminum foil). Let the samples cool down for 5 min, add 80 µL of distilled  H₂O and centrifuge the t…

Materials

Chemicals
N-acetylneuraminic acid	Sigma	A0812
N-glycolylneuraminic acid	Sigma	50644	1 mg aliquot should be sufficient
O-phenylenediamine	Sigma	694975
Sodium hydrogen sulfite	J&K Scientific Ltd	75234
HPLC Analysis
High-recovery HPLC vial	Agilent Technologies	#5188-2788
HPLC System	Shimadzu	Nexera
Fluorescence Detector for HPLC	Shimadzu	RF-20Axs
HPLC Column	Phenomenex	Hyperclone ODS	250 x 4.6 mm
LCMS-grade H2O	Merck Millipore	#WX00011
LCMS-grade Acetonitrile	Merck Millipore	#100029	Hypergrade
Ammonium hydroxide solution	Fluka	#44273	Puriss. p.a.