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

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 (F_Neu5Gc) from the fluorescence peak areas of the Neu5Ac (A_Neu5Ac) and Neu5Gc (A_Neu5Gc) as follows:
   F_Neu5Gc [%] = 100×A_Neu5Gc/(A_Neu5Ac+A_Neu5Gc). In case of the milk and liver samples from the homozygous Cmah knock-out mouse F_Neu5Gc should be 0%, whereas the values for F_Neu5Gc 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%.