Measurement of N-acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection.
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 alpha-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.
Ishida
J
J
Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Nakahara
T
T
Yamaguchi
M
M
D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008297:Male; D008875:Middle Aged; D019158:N-Acetylneuraminic Acid; D009369:Neoplasms / Q000097:blood / Q000652:urine; D009438:Neuraminic Acids / Q000032:analysis; D012794:Sialic Acids / Q000032:analysis* / Q000097:blood / Q000652:urine
D007202:Indicators and Reagents; D009438:Neuraminic Acids; D012794:Sialic Acids; C032592:N-glycolylneuraminic acid; D019158:N-Acetylneuraminic Acid