Use of a homologous internal standard for the quantification of the major metabolite of prostaglandin F 2α in human urine by multiple ion analysis

Abstract

A gas chromatographic-mass spectrometric method for quantitative determination of 9α,11α-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid, the major urinary metabolite of prostaglandin F 2α (PGF-M), was developed. The metabolite was analyzed as the dimethyl ester- O-methyloxime- bis-trimethylsilyl ether derivative. The internal standard consisted of a mixture of diethyl ester + monoethyl ester-δ-lactone of PGF-M. Those two species were converted to the 1-methyl-20-ethyl ester derivative during the analytical process. Linear standard curves were developed in the range 0 to 100 ng of injected prostaglandin. The method comprised extraction with Amberlite XAD-2, methylation, chromatography over octadecasilyl-silica, delactonization, remethylation, and chromatography over silicic acid and Lipidex-5000, followed by methoximation, trimethylsilylation, and instrumental analysis. Interassay coefficient of variation, for the analysis of four identical urine specimens, was 7% and intraassay coefficient of variation, when each sample was injected four times, ranged from 3.2 to 6.0%. Specificity, accuracy, and precision of the method were verified by recovery of the metabolite from two different urine pools. The recovery of authentic, underivatized PGF-M added to urine was 99.1 ± 2.4% (mean ± SE, N = 6). The plot of recovered versus added metabolite followed the equation y = 0.936 x + 25.8, with r = 0.9918.