Reproducibility of an assay to measure serum progesterone metabolites that may be related to breast cancer risk using liquid chromatography-tandem mass spectrometry.

Recent data suggest a novel role of progesterone in breast cancer etiology involving the progesterone metabolites 3α-dihydroprogesterone (3αHP), 5α-dihydroprogesterone (5αP), and 20α-dihydroprogesterone (20αHP). Accurate and precise measures of progesterone metabolites are needed for etiologic studies of hormonally related cancers. We have developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure five hormones, including progesterone, its precursor pregnenolone, and three progesterone metabolites, 5αP, 3αHP, and 20αHP. Hormone levels were measured in serum from 20 healthy volunteers (7 men, 5 premenopausal women, and 8 postmenopausal women). Two blinded, randomized aliquots per individual were assayed in each of four batches. The coefficients of variation (CV) and intraclass correlation coefficients (ICC) were calculated from the individual components of variance. The overall laboratory CVs were <3% and ICCs were uniformly high (>98%) for all hormones measured across sex/menopausal status groups. Our HPLC-MS/MS assay of progesterone metabolites demonstrated excellent sensitivity, laboratory reproducibility, and interindividual variation, suggesting that this serum assay is suitable for epidemiologic research. The high sensitivity of the assay, and thus the ability to quantify concentrations among postmenopausal women and men, further supports that this novel assay is suitable for studies of serum progesterone metabolite concentrations and risk of breast cancer or other hormonally related cancer.