Alvarez J G, Ludmir J
Department of Obstetrics and Gynecology, Beth Israel Hospital, Harvard Medical School, Boston, MA 02215.
J Chromatogr. 1993 May 19;615(1):142-7. doi: 10.1016/0378-4347(93)80300-s.
Analysis of the various lipids in amniotic fluid by multisample semiautomated quantitative high-performance thin-layer chromatography (HPTLC) is presented. Amniotic fluid (AF) lipids are extracted by liquid-liquid partition with chloroform-methanol (C-M) (final C-M-AF ratio of 4:2:1, v/v), 4-microliters aliquots of the C-M extract applied by way of an HPTLC autosampler device (Camag) to Whatman HP-K silica gel plates, and the lipids separated in one dimension using chloroform-ethanol-triethylamine-water (30:34:30:8, v/v) and hexane-diethyl ether (50:5, v/v) as the initial and final mobile phases, respectively. The plates were then stained with a 10% solution of copper sulfate in 8% phosphoric acid and placed in an oven with initial and final temperatures of 24 and 120 degrees C, respectively. This HPTLC system allowed resolution of cholesteryl palmitate, triglycerides, free fatty acid fraction, cholesterol, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. The stained chromatograms were scanned with a Shimadzu CS-9000U spectrodensitometer in the reflectance mode at 310 nm. The lower limit of detection was 20 ng/ml with linear detector response extended to 2 micrograms/ml. Use of this methodology allows the simultaneous analysis of the various lipids in AF for up to 50 samples in ca. 2 h. This method constitutes a sensitive, quantitative, reproducible, and high-capacity system for analysis of AF lipids.
本文介绍了采用多样品半自动定量高效薄层色谱法（HPTLC）对羊水内各种脂质进行分析的方法。羊水（AF）脂质通过氯仿 - 甲醇（C - M）液 - 液分配法提取（最终C - M - AF比例为4:2:1，v/v），将4微升C - M提取物通过HPTLC自动进样器装置（卡玛格）点样于沃特曼HP - K硅胶板上，脂质先以氯仿 - 乙醇 - 三乙胺 - 水（30:34:30:8，v/v）为流动相进行一维分离，最后以正己烷 - 乙醚（50:5，v/v）为流动相进行一维分离。然后将薄板用10%硫酸铜的8%磷酸溶液染色，并置于烘箱中，初始温度和最终温度分别为24℃和120℃。该HPTLC系统能够分离棕榈酸胆固醇酯、甘油三酯、游离脂肪酸组分、胆固醇、心磷脂、磷脂酰甘油、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰胆碱、鞘磷脂和溶血磷脂酰胆碱。用岛津CS - 9000U分光密度计在反射模式下于310 nm波长处扫描染色后的色谱图。检测下限为20 ng/ml，线性检测器响应范围扩展至2μg/ml。使用该方法可在约2小时内同时分析多达50个样品的羊水内各种脂质。该方法构成了一种用于分析羊水脂质的灵敏、定量、可重复且高通量的系统。
