Iizuka H, Yajima T
School of Pharmaceutical Sciences, Toho University, Chiba, Japan.
Biol Pharm Bull. 1993 Feb;16(2):103-6. doi: 10.1248/bpb.16.103.
It was observed in our laboratory that methoxyacetaldehyde induced strong fluorescence with L-tryptophan in the presence of NaNO2 at pH 2.75. The reaction mixture was heated at 80 degrees C for 15 min and the fluorescence was measured at the excitation wavelength, 253 nm and the emission, 450 nm. Amino acids other than L-tryptophan did not produce fluorescence under the conditions employed. A good linear working curve was observed between 25 pmol/50 microliters and 500 pmol/50 microliters of L-tryptophan. The limit of determination for L-tryptophan was 10 pmol/50 microliters. The coefficient of variation of the measurements (n = 5) was 1.5% for 250 pmol/50 microliters of L-tryptophan. The proposed method was successfully applied to the analyses of tryptophan in a pooled human serum. Total tryptophan was determined after deproteinization with trichloroacetic acid, and free tryptophan was determined using the ultrafiltrate of a pooled human serum. Analytical recoveries of total and free tryptophan were 98.8% and 99.1%, respectively. The fluorescent products of L-tryptophan and methoxyacetaldehyde were characterized as beta-carboline and 1-methoxymethyl-beta-carboline by the use of liquid chromatography-mass spectrometry (LC-MS) and HPLC.
在我们实验室观察到，在pH 2.75且存在亚硝酸钠的情况下，甲氧基乙醛与L-色氨酸可产生强烈荧光。反应混合物在80℃加热15分钟，然后在激发波长253nm和发射波长450nm处测量荧光。在所采用的条件下，除L-色氨酸外的其他氨基酸不产生荧光。在25pmol/50微升至500pmol/50微升的L-色氨酸之间观察到良好的线性工作曲线。L-色氨酸的测定限为10pmol/50微升。对于250pmol/50微升的L-色氨酸，测量的变异系数（n = 5）为1.5%。所提出的方法成功应用于混合人血清中色氨酸的分析。用三氯乙酸脱蛋白后测定总色氨酸，使用混合人血清的超滤液测定游离色氨酸。总色氨酸和游离色氨酸的分析回收率分别为98.8%和99.1%。通过液相色谱-质谱联用（LC-MS）和高效液相色谱（HPLC），L-色氨酸和甲氧基乙醛的荧光产物被鉴定为β-咔啉和1-甲氧基甲基-β-咔啉。
