Effect of blood and meconium on the determination of phospholipids in amniotic fluid using high pressure liquid chromatography

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Abstract
This paper the evaluation of the effects of blood and meconium on the determination of the phospholipids phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SP) and lysolecithin (LL) in amniotic fluid. Phospholipids were analyzed by a new method using high pressure liquid chromatography (HPLC), which is based on the procedure of Briand et al. (1). The method was extended for quantitative determination with lysolecithin as internal standard. The HPLC equipment consisted of two pumps, an HPLC programmer, an HPLC oven, a UV detector and an integrator. The chromatographic separation was achieved on a 25 cm DIOL-column and a 6 cm guard column packed with silica SI 60. The oven temperature was 55.degree. C and the detector wave length was 201 nm. The chromatographic mobile phase was composed of two solvents, acetonitrile and water. A solvent gradient was run from 2.4% water to 15% water between 5 and 13 minutes. Phospholipids were extracted according to the procedure of Gluck (8). Before extraction 40 .mu.g of LL as internal standard were added to 2.0 ml amniotic fluid. In a standard solution the phospholipids PE, PC, SP and LL were baseline separated, in the case of PG and PI a perpendicular division of the peaks was necessary. To evaluate the effect of contamination by blood and meconium, various amounts of blood and meconium were added to uncontaminated amniotic fluid samples. Contamination by blood caused a rise of the concentrations of PC, PE and SP up to tenfold. PG and PI concentration were not affected by blood staining. HPLC analysis proved that blood contains only PE, PC and SP in detectable amounts, but no Pg and PI. In specimens with blood contaminants, only PG and PI are reliable indicators of fetal lung maturity. The concentrations of PG PI and PE were not affected by meconium staining. PC and SP values showed an ascending drift up to a maximum rise of 20% resp. 10%. HPLC analysis showed that meconium contains small amounts of PC and SP, but not PG, PI and PE. PC and SP determinations are therefore practicable only in slightly meconium stained specimens.