$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
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 tubes at 14,000 x g for 1 min.
- 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
- Analyze the samples using a standard HPLC system connected to an online fluorescence detector.
- Use a reversed phase C18 column with the standard dimensions of 250 mm length and 4.6 mm diameter for the analysis.
- 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:
- Add 46 g of formic acid to 800 mL of LCMS-grade H₂O.
- Adjust the pH to 4.5 by dropwise adding ammonium hydroxide solution (puriss. p.a.).
- 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.
- For solvent B, use LCMS-grade acetonitrile.
- Separate the sialic acids derivatives at a 1 mL/min flow rate with the following gradient elution:
- Start by adding 10% of solvent B mixed to solvent A.
- From 0 min to 15 min, gradually increase the proportion of solvent B with a linear gradient to 60%.
- 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.
- 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.
- Re-equilibrate HPLC column to the starting conditions of 10% B between 19 and 24 min.
- Inject 50 µL of sample into the HPLC system.
- 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).
- 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%.