SEMIAUTOMATED MULTISAMPLE ANALYSIS OF AMNIOTIC-FLUID LIPIDS BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY REFLECTANCE SPECTRODENSITOMETRY

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-mul aliquots of the C-M ''tract 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 mug/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.