Determination of prostaglandins in human seminal fluid by solid-phase extraction, pyridinium dichromate derivatization and high-performance liquid chromatography

Prostaglandins of the E type (PGEs) in human seminal fluid have been determined by reversed-phase high-performance chromatography on a C18 column and ultraviolet detection at 230 nm after solid-phase extraction (C18) and oxidation to the corresponding 15-oxoprostaglandin derivatives by pyridinium dichromate in acetonitrile. Under optimized conditions, PGEs from 10-ml seminal samples were extracted into 4 ml of methyl formate with high recoveries (estimated at greater than 95%) and subsequently separated under mild chromatographic conditions (0.5 mM formic acid-acetonitrile, apparent pH 3.8). Comparable analytical sensitivities were obtained with detection at 230 nm with a conventional deuterium lamp spectrophotometer and a photometer equipped with a cadmium emission source, while with a diode-array spectrophotometer, signal-to-noise ratios were reduced with factors between 4.4 and 3.1, depending on the spectral bandwidth of the instrument. Theoretical aspects of signal-to-noise optimization of ultraviolet detectors are discussed. The stability of dilute standard solutions of PGE2 and PGD2 was measured, showing solutions in dichloromethane at 20 degrees C to be as stable as acetonitrile solutions at 5 degrees C over a period of thirty days. Absolute ethanol and acetonitrile were equally suited as solvents.