Yoon H T, Yoo H S, Shin B K, Lee W J, Kim H M, Hong S P, Moon D C, Lee Y M
College of Pharmacy, Chungbuk National University, Chongju, Korea.
Arch Pharm Res. 1999 Jun;22(3):294-9. doi: 10.1007/BF02976365.
Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphingosine and sphinganine, was investigated to obtain high fluorescent detectability. In order to improve the fluorescent yield, we investigated the optimal solubility of sphingoid bases for five pre-incubation solvents by incorporating the heating procedure before OPA derivatization. The pre-incubation in ethanol prominently increased the fluorescent peak height of OPA derivative for each sphingoid bases in high performance liquid chromatography. About ten-fold increase of detectability was archived by pre-incubating lipid extracts pellets in ethanol at 60 degrees C for 30 min. Optimal derivatization was performed in 30 min at ambient temperature and the fluorescent intensity of OPA derivative was stable for two weeks at 4 degrees C. The detection limit of sphingosine was 0.1 pmol as injected amount. This method was applied to the determination of cellular sphingosine and sphinganine in various human lung cancer cells. This OPA procedure was prospective to be useful for quantitating the amount of sphingoid bases in other cancer cells.
研究了鞘氨醇碱、鞘氨醇和二氢鞘氨醇的柱前邻苯二甲醛（OPA）标记方法，以获得高荧光检测能力。为了提高荧光产率，我们通过在OPA衍生化之前加入加热步骤，研究了五种预孵育溶剂中鞘氨醇碱的最佳溶解度。在乙醇中预孵育显著提高了高效液相色谱中每种鞘氨醇碱的OPA衍生物的荧光峰高。通过在60℃的乙醇中预孵育脂质提取物沉淀30分钟，检测能力提高了约10倍。在室温下30分钟内进行最佳衍生化，OPA衍生物的荧光强度在4℃下可稳定两周。鞘氨醇的检测限为0.1皮摩尔（注入量）。该方法应用于各种人肺癌细胞中细胞鞘氨醇和二氢鞘氨醇的测定。这种OPA方法有望用于定量其他癌细胞中鞘氨醇碱的含量。
