Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver
Published: April 30, 2023
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…
Disclosures
The authors have nothing to disclose.
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. |
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Cite This Article
Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver. J. Vis. Exp. (Pending Publication), e21100, doi: (2023).