Validation of routine methods for serum progesterone determination using mass fragmentography

Abstract

A mass fragmentographic reference method for determination of serum progesterone is described. A fixed amount of [4- 14C] progesterone (usually 7.5 ng) is added to a fixed amount of serum (usually 1 ml) and extracted with hexane. The extract is purified by means of thin-layer chromatography. The purified progesterone is converted into the dienol heptafluorobutyrate derivative by treatment with heptafluorobutyric acid anhydride. The amount of unlabeled progesterone is determined from the ratio between the recordings at m/ e 510 and m/ e 512 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). The two ions used correspond to the molecular peak in the mass spectrum of the dienol heptafluorobutyrate derivative of unlabeled and labeled progesterone, respectively. The relative standard deviation of the method was 3.7% provided that serum progesterone concentration was in the range 10–70 nM. Two radioimmunoassay techniques with commercial kits were compared with the mass fragmentographic method. The correlation coefficient obtained was 0.93 and 0.7 6 respectively, and the regression coefficient 0.93 and 1.05, respectively.