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Measurement of N ‐acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection
Author(s) -
Ishida Junichi,
Nakahara Toshihiro,
Yamaguchi Masatoshi
Publication year - 1992
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130060308
Subject(s) - chemistry , chemiluminescence , chromatography , detection limit , urine , reagent , derivatization , hydrogen peroxide , elution , hydrochloric acid , n acetylneuraminic acid , high performance liquid chromatography , sialic acid , biochemistry , organic chemistry
A highly sensitive method for the determination of N ‐acetylneuraminic acid in human serum and urine is investigated. This method employs high performance liquid chromatography with chemiluminescence detection. N ‐Acetylneuraminic acid, released by hydrochloric acid hydrolysis of serum and urine, and N ‐glycolylneuraminic acid (internal standard) are converted into chemiluminescent derivatives with 4,5‐diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for α‐keto acids. The derivatives are separated within 35 min on a reversed phase column, TSKgel ODS‐120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for N ‐acetylneuraminic acid is 9 fmol (signal‐to‐noise ratio = 3). This sensitivity permits precise determination of N ‐acetylneuraminic acid in 10 nL of serum or 50 nL of urine. The method is applied to the determination of the N ‐acetylneuraminic acid in human sera from normal subjects and cancer patients and in normal urine.