Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-13C3 N-Acetyl-d-neuraminic Acid (13C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366 → 330 and 369 → 333 for Neu5Ac and 13C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 μM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 μM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

► When sialic acid is assayed by reversed-phase LC its short retention time leads to important ion suppression effects.
► We butylated sialic acid to improve its analytical separation.
► Butylated sialic acid allowed a more sensitive detection in positive mode.
► Stable isotope of sialic acid was used as an internal standard to minimize matrix effect.