Low levels of prostaglandins in human milk after purification by high performance liquid chromatography

In order to validate methodology for quantifying arachidonic acid metabolites in human milk, we measured five prostaglandins (PGs) using radioimmunoassay (RIA): PGE2, PGF2 alpha, thromboxane B2 (TXB2), 6-keto-PGF1 alpha (6KF1 alpha) and 13,14-dihydro-15-keto-PGF2 alpha (DHKF2 alpha). RIA was performed after extraction with C18 Sep-Pak cartridge and/or further purification with reverse-phase high performance liquid chromatography (rp-HPLC). Recovery was monitored throughout the purification steps. Confirming findings of previously reported studies, we found PGE and the PGF2 alpha metabolite, DHKF2 alpha to be the major PGs in extracted human milk. After HPLC purification, however, no predominant PG was found, and the PG levels, especially those of PGE2 and DHKF2 alpha, were considerably lower than levels in the extracted sample. A noteworthy finding was the presence of immunoreactive substances in HPLC fractions not within the area of the PG peak; this suggests that the decreased levels of PGs in human milk measured by RIA after rp-HPLC are due to the elimination of the nonspecific immunoreactive materials present in the sample.