Sialic acids are one of the important constituents of glycoconjugates in microorganisms and a wide range of higher animals. Many sialic acids are bound to sugar chains through alpha-ketoside linkages such as alpha-2,3, alpha-2,6 and alpha-2,8. They are deeply involved in cell-to-cell recognition, blood coagulation, fertilization and other biological events. Conventional methods such as the resorcinol assay and thiobarbituric acid test involve several complex procedures.

However, the HPLC-based sialic acid fluorescence labeling technique using 1,2-diamino-4,5-methyleneoxybenzene (DMB) is a relatively simple and highly sensitive quantitative method. In this method, free sialic acids are analyzed by reverse phase HPLC (PALPAK Type R) after labeling by DMB. Not only Neu5Ac but also NeuGc, KDN and their O-acetyl derivatives can be analyzed separately by this method, and the detection limit is <57 fmol. Combining this DMB method with the pyridylamino (PA) derivatization method allows sialoglycoconjugates to be successfully analyzed with extremely high sensitivity.

Procedure

1. Set samples containing free sialic acids (ranging from 5 pmol to 5 nmol into screw capped 1.5 mL tubes. Note: The sample volume must be kept under 50 µL. Since acidic conditions are required for the following reaction, the pH must be kept neutral or acidic.
2. Prepare a mixed solution, with a ratio of Reagent 1:Reagent 2: H₂O of 1:5:4. Add 200 µL of this mixed solution to the samples, stir and allow reaction to proceed for 2.5 hours at 50°C, protected from light. Note: This mixed solution is stable at least for a week at 4°C.
3. Cool on ice to terminate the reaction.
4. Inject 10 µL of the reaction mixture into a HPLC column. Quantities of sialic acids within the sample can be calculated from the calibration curve based on the peak height obtained with the defined sialic acid standard.