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-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-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 (13)C-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).
Laboratoire de Biochimie Hormonologie, Hôpital Robert Debré, Paris, France.
Abdellah
A
Tebani
Dimitri
D
Schlemmer
Apolline
A
Imbard
Odile
O
Rigal
Dominique
D
Porquet
Jean-François
JF
Benoist
D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003404:Creatinine / Q000652:urine; D004355:Drug Stability; D006801:Humans; D016018:Least-Squares Analysis; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D029461:Sialic Acid Storage Disease / Q000652:urine; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002247:Carbon Isotopes; D003404:Creatinine; D019158:N-Acetylneuraminic Acid