Progesterone Metabolism in Serum

Significance of the Topic

Progesterone and its downstream metabolites are key regulators in female physiology and are implicated in hormone replacement therapies and cancer development, particularly breast cancer. Accurate profiling of these steroids in serum supports clinical research, therapeutic monitoring, and risk assessment.

Objectives and Study Overview

This application note presents a validated LC–MS/MS approach to quantify progesterone and six related metabolites in human serum. The primary aims were to achieve chromatographic separation of structural isomers, establish sensitive detection limits, and demonstrate reproducible quantification in male and postmenopausal female samples.

Methodology and Instrumentation

The workflow combines liquid–liquid extraction, chemical derivatization, and tandem mass spectrometry.

• Sample Preparation: 400 µL serum spiked with 13C-labeled internal standard, extracted twice with tert-butyl methyl ether, evaporated, derivatized with hydroxylamine at 90 °C, re-extracted, and reconstituted in 70/30 water/acetonitrile.
• Chromatography: Agilent 1290 Infinity II system with BEH C18 column (100 × 2.1 mm, 1.7 µm) at 30 °C. Gradient elution from 20% to 95% acetonitrile with 0.1% formic acid over 8 min at 300 µL/min.
• Mass Spectrometry: Agilent 6495 triple quadrupole operated in positive ionization mode. Optimized gas settings, capillary voltage, and multiple reaction monitoring (MRM) transitions for each analyte ensured selective detection.

Results and Discussion

The method achieved baseline separation of seven steroids, including deoxycorticosterone, 20α-, 5α-, and 3α-dihydroprogesterone, allopregnanolone, pregnanolone, and progesterone. Limits of quantification ranged from 0.8 to 4.1 ng/L. Intra- and inter-assay precision were below 10% for all analytes. Serum profiling in men (n = 64) and postmenopausal women (n = 54) yielded median progesterone levels of ~48 ng/L and ~28 ng/L, respectively, and consistent metabolite ratios in line with literature.

Benefits and Practical Applications

This LC–MS/MS protocol offers:

• High sensitivity and specificity for low-abundance steroids.
• Robust quantification across a clinical concentration range.
• Applicability to research in endocrinology, oncology, and therapeutic monitoring.

Future Trends and Applications

Advances may include automation of sample preparation, expansion to additional steroid panels, integration with high-resolution MS, and application to large-scale epidemiological studies to refine hormone-cancer risk models.

Conclusion

The described LC–MS/MS method provides a reliable, validated approach for serum progesterone metabolite profiling. It supports diverse clinical research needs by combining efficient sample processing with sensitive detection and reproducible quantification.

References

1. Trabert B. et al. Progesterone and Breast Cancer. Endocr. Rev. 41(2), 320–344 (2020).
2. Trabert B. et al. Association of Circulating Progesterone With Breast Cancer Risk Among Postmenopausal Women. JAMA Netw. Open 3(4), e203645 (2020).
3. Wiebe J.P. Progesterone Metabolites in Breast Cancer. Endocr. Relat. Cancer 13(3), 717–738 (2006).
4. Trabert B. et al. Reproducibility of an Assay to Measure Serum Progesterone Metabolites That May Be Related to Breast Cancer Risk Using Liquid Chromatography–Tandem Mass Spectrometry. Horm. Mol. Biol. Clin. Investig. 23(3), 79–84 (2015).
5. Stanczyk F.Z. et al. Progestogens Used in Postmenopausal Hormone Therapy: Differences in Their Pharmacological Properties, Intracellular Actions, and Clinical Effects. Endocr. Rev. 34(2), 171–208 (2013).