Essig S, Kovar K A
University of Tübingen, Germany.
J AOAC Int. 2001 Jul-Aug;84(4):1283-6.
This paper describes a convenient and practice method for quantitation of surfactant phospholipids (1,2-dipalmitoyl-3-sn-phosphatidyl choline [DPPC] and 1-palmitayl-2-oleyl-3-sn-phosphatidyl glycerol [POPG]) in a recombinant surfactant lyophile (Venticute) by high-performance, thin-layer chromatography (HPTLC) with video densitometry. DPPC and POPG were extracted from Venticute-lyophile using methanol. Separation from the other active ingredients and excipients was accomplished by HPTLC on silica gel F254 plates with a mixture of chloroform, methanol, glacial acetic acid, and water as development solvent. Postchromatographic derivatization by dipping in copper sulphate/phosphoric acid reagent and subsequent heating shows grey-brown bands on a light blue background. These were detected with the video densitometer in the VIS range, and with scanning densitometry at 365 nm. Linear calibration in a working range of 0.7-1.3 microg DPPC and 0.35-0.65 microg POPG was demonstrated by integrating the area under the peaks. Good results were obtained with recovery experiments. When compared to classical slit scanning densitometry, video densitometry represents a fast alternative to quantitate thin-layer chromatograms in surfactant phospholipid analysis.
本文描述了一种通过高效薄层色谱（HPTLC）结合视频光密度法对重组表面活性剂冻干剂（Venticute）中表面活性剂磷脂（1,2 - 二棕榈酰 - 3 - sn - 磷脂酰胆碱[DPPC]和1 - 棕榈酰 - 2 - 油酰 - 3 - sn - 磷脂酰甘油[POPG]）进行定量的简便实用方法。使用甲醇从Venticute冻干剂中提取DPPC和POPG。在硅胶F254板上，以氯仿、甲醇、冰醋酸和水的混合物作为展开溶剂，通过HPTLC实现与其他活性成分和辅料的分离。通过浸入硫酸铜/磷酸试剂进行色谱后衍生化，随后加热，在浅蓝色背景上显示出灰棕色条带。在可见光范围内用视频光密度计检测这些条带，并在365 nm处进行扫描光密度测定。通过积分峰下面积，在0.7 - 1.3μg DPPC和0.35 - 0.65μg POPG的工作范围内证明了线性校准。回收率实验取得了良好结果。与经典的狭缝扫描光密度法相比，视频光密度法是表面活性剂磷脂分析中定量薄层色谱图的一种快速替代方法。
