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Background Current analytical routine methods lack the sensitivity to monitor plasma estrogen levels in breast cancer patients treated with aromatase inhibitors. Such monitoring is warranted for premenopausal patients treated with an aromatase inhibitor and an LH-releasing hormone analogue in particular. Therefore, we aimed to develop a routine tandem mass spectroscopy combined with liquid chromatography (LC-MS/MS) method for estradiol (E2) and estrone (E1) for use in the sub-picomolar range. Method Calibrators, quality controls (QC), or serum samples were spiked with isotope-labeled internal standard and purified by liquid-liquid extraction. The reconstituted extracts were analyzed by LC-MS/MS in negative electrospray ionization mode. QCs at 6 levels made from pooled patient sera were used to validate the accuracy, sensitivity, and precision of the method. Results We achieved limits of quantification of 0.6 pmol/L (0.16 pg/mL) for E2 and 0.3 pmol/L (0.07 pg/mL) for E1. The coefficient of variation was below 9.0% at all QC levels for E2 (range, 1.7-153 pmol/L), and below 7.8% for E1 (range, 1.7-143 pmol/L). The method is traceable to the E2 reference standard BCR576. Reference ranges for E2 and E1 in healthy, postmenopausal women were obtained, for E2: 3.8 to 36 pmol/L, for E1: 22 to 122 pmol/L. We measured and confirmed ultra-low E2 and E1 concentrations in sera from patients on the aromatase inhibitors letrozole or exemestane. Conclusion This ultrasensitive LC-MS/MS method is suitable for routine assessment of serum E1 and E2 levels in breast cancer patients during estrogen suppression therapy. The method satisfies all requirements for measurement of E2 in the clinical setting as stated by the Endocrine Society in 2013. Precis We report an ultrasensitive LCMS/MS routine assay that measures pretreatment and suppressed levels of estradiol/estrone during aromatase inhibitor treatment of postmenopausal breast cancer patients.

An Ultrasensitive Routine LC-MS/MS Method for Estradiol and Estrone in the Clinically Relevant Sub-Picomolar Range