Determination of free N-acetylneuraminic acid by ultrafiltration and liquid chromatography in the serum of normal and cancer patients

An ultrafiltration technique coupled with liquid chromatography and spectrofluorimetric (λ(ex) = 373 nm, λ(em) = 448 nm) detection is described for rapidly assaying free N-acetylneuraminic acid (Neu5Ac) in the serum of normal and cancer patients. Free Neu5Ac was obtained from serum after ultrafiltration hy centrifugation through an Amicon Centrifree micropartition filter which has a relative molecular mass cut-off of 1000 daltons. Aspects of the ultrafiltration procedure include the extent of non-specific binding of Neu5Ac to the ultrafilters, precision of recovery and the effect of temperature. Non-specific losses of Neu5Ac in serum to the ultrafilter system were low (recovery > 91.2%). The detection limit (S/N = 3) = 2 ng/ml. For free Neu5Ac at 180-1421 ng ml-1, the intra- and inter-day C.V.s for the ultrafiltration method were ≤ 2.8% and ≤ 3.9%, respectively. Free Neu5Ac concentrations measured by this method were compared to those measured by the acetonitrile procedure. The results showed an average value of 245 ± 90 ng ml-1for 30 serum samples of normal subjects. The average value for 20 lung cancer patients was found to be 996 ± 310 ng ml-1.