A new method for the determination of the major metabolite of prostaglandin F2alpha in human urine based on stable isotope dilution and gas chromatography-mass spectrometry.

Studies on the biological fate in man of primary prostaglandins of the E and F series have shown that metabolic conversion takes place into a series of side-chain degradation products that are excreted in urine (Samuelsson, 1973). The major metabolite of prostaglandin FzQ in the urine is 5a,7a-dihydroxy-ll-oxotetranorprostane-l,16-dioic acid (referred to below as ‘prostaglandin F metabolite’), quantitative determination of which may be used to study the effect of drugs or different physiological conditions on prostaglandin F2. turnover in uiuo (Hamberg, 1974; Samuelsson, 1973). In connection with our investigations into the role of prostaglandin F,, in certain pathological disorders, we have developed a highly specific and sensitive assay for prostaglandin F metabolite, based on stable isotope dilution and combined g.1.c.-mass spectrometry. The internal standard used in this assay is a ZH-labelled analogue of prostaglandin F metabolite, in which the ,H atoms occupy positions 4/3,6a and 68 in the cyclopentane ring. This compound was isolated from the urine of a female Rhesus monkey which had received an intravenous infusion of [88,10a,lOB-*H~, 98-3H]prostaglandin Fz. (14.5mg). (This labelled precursor was prepared from prostaglandin Ez by base-catalysed equilibration in [hydr~xy-~H]carbitol, followed by reduction with NaB3H4, and chromatographic separation of the prostaglandin F isomers obtained.) Purification of the labelled metabolite (yield Img), as its methyl ester derivative was achieved by means of liquid-gel partition chromatography (Nystrom & Sjovall, 1973; Brash & Jones, 1974). Fig. 1 summarizes the sequence of steps in the assay procedure. To a portion (10mI) of a 24h urine collection is added the methyl ester of [2H3]prostaglandin F metabolite (200ng) followed by ~ M N ~ O H (2ml). Hydrolysis of this methyl ester and of the 6lactone form of the endogenous metabolite is allowed to proceed at ambient temperature overnight, resulting in equilibration of the two molecular species of prostaglandin F metabolite as the salt of the dioic acid. The urine sample is then acidified to pH3 and the prostaglandins are extracted on a small column (2g) of Amberlite XAD-2. Elution of the column with methanol, and esterification of the material so obtained with 2 % methanolic tetramethylammonium hydroxide and methyl iodide (Greeley, 1974), affords the metabolite as a mixture of its methyl ester and b-lactone methyl ester derivatives. These compounds are then converted into the 16-monomethyl ester of prostaglandin F meta-