Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization.
N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected].
Fukushima
Takeshi
T
Division of Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan. fukushima@u-shizuoka-ken.ac.jp
Arai
Kotaro
K
Tomiya
Masayuki
M
Mitsuhashi
Shogo
S
Sasaki
Tsukasa
T
Santa
Tomofumi
T
Imai
Kazuhiro
K
Toyo'oka
Toshimasa
T
D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis; D001923:Brain Chemistry; D054022:Cerebrum / Q000737:chemistry*; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013050:Spectrometry, Fluorescence
D015415:Biomarkers; D005456:Fluorescent Dyes; D001224:Aspartic Acid; C000179:N-acetylaspartate