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 semiau tomated quantitative high-performance thin-layer chromatography (HPTLC ) is presented. Amniotic fluid (AF) lipids are extracted by liquid-liq uid 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 HPTL C autosampler device (Camag) to Whatman HP-K silica gel plates, and th e lipids separated in one dimension using chloroform-ethanol-triethyla mine-water (30:34:30:8, v/v) and hexane-diethyl ether (50:5, v/v) as t he initial and final mobile phases, respectively. The plates were then stained with a 10% solution of copper sulfate in 8% phosphoric acid a nd placed in an oven with initial and final temperatures of 24 and 120 -degrees-C, respectively. This HPTLC system allowed resolution of chol esteryl palmitate, triglycerides, free fatty acid fraction, cholestero l, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, phosph atidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin , and lysophosphatidylcholine. The stained chromatograms were scanned with a Shimadzu CS-9000U spectrodensitometer in the reflectance mode a t 310 nm. The lower limit of detection was 20 ng/ml with linear detect or response extended to 2 mug/ml. Use of this methodology allows the s imultaneous analysis of the various lipids in AF for up to 50 samples in ca. 2 h. This method constitutes a sensitive, quantitative, reprodu cible, and high-capacity system for analysis of AF lipids.