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

  1. 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.
  2. 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).
  3. Let the samples cool down for 5 min, add 80 µL of distilled  H₂O and centrifuge the tubes at 14,000 x g for 1 min.
  4. Transfer 80 µL from the supernatant into a 300 µL high-recovery HPLC vial. The derivatized sialic acid samples can be stored at 4-6 °C for up to one week.

2. HPLC Analysis of Sialic Acid Derivatives

  1. Analyze the samples using a standard HPLC system connected to an online fluorescence detector.
  2. Use a reversed phase C18 column with the standard dimensions of 250 mm length and 4.6 mm diameter for the analysis.
  3. Prepare solvent A by diluting 200 mL of stock solution with 800 mL of LCMS-grade water (LCMS – liquid chromatography mass-spectrometry). The stock solution itself can be prepared as follows:
    1. Add 46 g of formic acid to 800 mL of LCMS-grade H₂O.
    2. Adjust the pH to 4.5 by dropwise adding ammonium hydroxide solution (puriss. p.a.).
    3. Transfer the solvent to a measuring cylinder and fill up to 1,000 mL with LCMS-grade  H₂O. This stock solution can be stored at 4-6 °C for up to 3 months.
  4. For solvent B, use LCMS-grade acetonitrile.
  5. Separate the sialic acids derivatives at a 1 mL/min flow rate with the following gradient elution:
    1. Start by adding 10% of solvent B mixed to solvent A.
    2. From 0 min to 15 min, gradually increase the proportion of solvent B with a linear gradient to 60%.
    3. From 15 min to 16 min, rapidly increase the proportion of solvent B with a linear gradient from 60% to 90%. This initiates the washing of the HPLC column.
    4. To further wash the HPLC column, keep the level of solvent B at 90% between 16 min and 18 min before gradually reducing the level of solvent B again to 10% between 18 min and 19 min.
    5. Re-equilibrate HPLC column to the starting conditions of 10% B between 19 and 24 min.
  6. Inject 50 µL of sample into the HPLC system.
  7. Monitor eluents using the fluorescence detector excitation/emission wavelengths of 373/448 nm, and the derivatized sialic acids can be expected at the approximate retention times of 9 min (for Neu5Gc-OPD) and 10 min (for Neu5Ac-OPD).
  8. Calculate the relative amount of Neu5Gc (FNeu5Gc) from the fluorescence peak areas of the Neu5Ac (ANeu5Ac) and Neu5Gc (ANeu5Gc) as follows:
    FNeu5Gc [%] = 100×ANeu5Gc/(ANeu5Ac+ANeu5Gc). In case of the milk and liver samples from the homozygous Cmah knock-out mouse FNeu5Gc should be 0%, whereas the values for FNeu5Gc of heterozygous and wild-type mice may vary widely depending on mouse age and tissue type (between 2% and >90%) with expected error margins of ±12%.

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).

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