Semiautomated multisample analysis of amniotic fluid lipids by high-performance thin-layer chromatography-reflectance...

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.