Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Abstract

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.

Confirmation of ovulation from urinary progesterone analysis: assessment of two automated assay platforms

“…Time-resolved fluorimetry requires more specialised equipment than the competitive TMB-based ELISA, hence it is likely this technique will be predominantly utilized by specialised reproductive laboratories. Liquid chromatography and tandem mass spectroscopy represent an accurate alternative 21 , however the cost is likely to be prohibitive in the general laboratory. Autoanalyzers such as those tested here are in widespread use for plasma/serum in general biochemistry laboratories and using them for confirmation of ovulation, where available, would be of great practical value, reducing direct and indirect costs and improving efficiency.…”

Penetapan Retention Time Progesteron Dalam Pelarut Eluen Phase Mobile Menggunakan High Performance Liquid Chromatography

“…adalah hormon steroid yang disintesis terutama di ovarium, plasenta dan kelenjar adrenal (Graham JD et al, 1997;Sinreih M et al, 2015). Progesteron banyak digunakan untuk terapi hormon oleh karena itu perlu dibentuk metode yang sederhana dan akurat untuk identifikasi dan penentuan kuantitatif dalam obatobatan (Maliwal et al, 2009).…”

Metabolic flux analysis: a comprehensive review on sample preparation, analytical techniques, data analysis, computational modelling, and main application areas

“…6 MFA can be now supported by the use of stable isotopes detectable as tracers by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. 7 In 13 C-MFA one 13 C-labelled (e.g. [1,[2][3][4][5][6][7][8][9][10][11][12][13] C] glucose; [1,[6][7][8][9][10][11][12][13] C] glucose; uniformly labelled [U- 13 C] glucose; 13 C-CO 2 ; 13 C-NaHCO 3 , etc.)…”

“…7 In 13 C-MFA one 13 C-labelled (e.g. [1,[2][3][4][5][6][7][8][9][10][11][12][13] C] glucose; [1,[6][7][8][9][10][11][12][13] C] glucose; uniformly labelled [U- 13 C] glucose; 13 C-CO 2 ; 13 C-NaHCO 3 , etc.) or multiple singly labelled substrates (COMPLETE-MFA) 8 are used as the carbon source for cell growth.…”