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.

Fig 1. Proposed progesterone metabolism in human endometrium.

Progesterone is metabolized by 20-ketosteroid reductases and 5α-reductases, to form (20S/R)-pregn-4-ene-3α,20-diol and 5α-pregnane-3,20-dione. Reductions of the 3-keto and 20-keto groups are catalysed by the aldo-keto reductases AKR1C1-AKR1C3. Here, AKR1C1-AKR1C3 can act on the 20-keto group of pregn-4-enes, and they can reduce both 3-keto and 20-keto groups of 5α-pregnanes. The progesterone (20S)-pregn-4-ene-3α,20-diol formed by the AKR1C enzymes can be oxidized back to progesterone by the 20α-hydroxysteroid dehydrogenase activity of 17β-hydroxysteroid dehydrogenase type 2 (HSD17B2). The formation of 5α-pregnanes is irreversible and is catalysed by 5α-reductases types 1 (SRD5A1) and 2 (SRD5A2) [–37].

Fig 2. Reaction scheme and structural formulae of the pregn-4-ene-metabolites.

Reagents and conditions: (a) Al(O-i-Pr)₃, 2-propanol, reflux.

Fig 3. Representative chromatogram showing separation of progesterone and its metabolites.

Representative extracted- ion chromatogram of a 100 ng/mL standard mixture of progesterone and its metabolites.

Fig 4. Representative calibration curves in solvent.

A) (20S)-pregn-4-ene-3α,20-diol (r = 0.9999, y = 0.00015x). B) (20S)-5α-pregnane-3α,20-diol (r = 0.9998, y = 0.00014). Curves are constructed out of three measurements for each concentration.

Fig 5. Representative calibration curves in matrix.

A) (20S)-pregn-4-ene-3α,20-diol (r = 0.9985, y = 0.00015x). B) (20S)-5α-pregnane-3α,20-diol ((r = 0.9997, y = 0.00015). Curves are constructed out of three measurements for each concentration.

Fig 6. Progesterone metabolites formed in the HES cell line.

Histogram representing the percentages of progesterone and its most abundant metabolites detected at different times. The scheme of the proposed metabolism is shown below. The main pathway is marked in bold. First, the 20-keto group of progesterone is reduced to form (20S)-20-hydroxy-pregn-4-ene-3-one, which is reduced at position C5 to form (20S)-20-hydroxy-5α-pregnane-3-one. Then (20S)-20-hydroxy-5α-pregnane-3-one is further metabolized at the 3-keto group, to form mainly (20S)-5α-pregnane-3α,20-diol, and also (20S)-5α-pregnane-3β,20-diol to a lesser extent.