通过高效液相色谱法分析生物组织中氨基酸和生物胺的邻苯二甲醛/乙硫醇/芴甲氧羰酰氯衍生物。脱蛋白研究。
Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography. A deproteinization study.
作者信息
Korös A, Hanczkó R, Jámbor A, Qian Y, Perl A, Molnár-Perl I
机构信息
Institute of Chemistry, Department of Analytical Chemistry, L. Eötvös University, Budapest, Hungary.
出版信息
J Chromatogr A. 2007 May 11;1149(1):46-55. doi: 10.1016/j.chroma.2006.11.018. Epub 2006 Dec 4.
The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of < or =2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.
为了通过高效液相色谱法（HPLC）对鸟氨酸、赖氨酸、腐胺、尸胺、1,7-二氨基庚烷、亚精胺和精胺进行定量分析，对从生物组织中提取这些物质作为其邻苯二甲醛/乙硫醇/芴甲氧羰酰氯（OPA/ET/FMOC）衍生物的方法进行了优化。在应用高氯酸脱蛋白时，采用了两种方法：（i）脱蛋白后用氢氧化钾中和并冻干，以及（ii）脱蛋白后不进行中和和冻干。中和和冻干导致游离生物胺损失。对高氯酸脱蛋白样品（模型溶液和组织）的上清液中作为OPA/ET/FMOC衍生物的生物组织中鸟氨酸（Orn）、赖氨酸（Lys）、腐胺（Put）、尸胺（Cad）、1,7-二氨基庚烷（Dah）、亚精胺（Spd）和精胺（Spm）含量进行HPLC分析，平均相对标准偏差（RSD）≤2.6%，包括样品处理和色谱分析的回收率。
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