MSACL 2019 EU Abstract

An Ultrasensitive High-Throughput LCMS/MS-Method for Estradiol and Estrone in the Sub-Picomolar Range

Bjørn-Erik Bertelsen (1), Ralf Kellmann (1), Kristin Viste (1), Hans Petter Eikesdal (2,3), Per Eystein Lønning (2,3), Jørn V. Sagen (1,4), Bjørg Almås (1)
(1) Hormone Laboratory, Haukeland University Hospital, Bergen, Norway (2) Department of Oncology Haukeland University Hospital, Bergen, Norway (3) Department of Clinical medicine (4) Department of Clinical science

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Bjørn-Erik Bertelsen (Presenter) Hormone Laboratory, Haukeland University Hospital

Presenter Bio: Method Developer at Haukeland University Hospital, Hormone Laboratory.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Background: We intended to develop a high-throughput LCMS-method to measure sub-picomolar levels of estradiol (E2) and estrone (E1). The method should be able to differentiate between pretreatment and suppressed blood levels of E2 and E1 in postmenopausal breast cancer patients undergoing aromatase inhibitor (AI) treatment, and should be well applicable in a routine lab.

Method: Our method development strategy involved extensive optimization of a less sensitive (LOQ 13 pmol/L) LC-MS/MS routine method for E2 currently in use in our lab. Systematic approach to the LLE sample preparation step, including solvents and mixing conditions resulted in optimal outcomes with regard to extraction recovery and sample purity. In brief: Serum or plasma samples were spiked with isotopically labeled internal standard and extracted in 96-well plates with hexane:methyl tert-butyl ether in an automated procedure (Hamilton Star Line). Reconstituted extracts were analyzed using LC-MS/MS (SCIEX API6500+) in negative ESI mode. Certified reference materials (CRM), and quality controls (QC) made from pooled patient sera were used to validate sensitivity, accuracy and precision of the method.

Results: Sample preparation, including LLE, evaporation and reconstitution is performed in about 4 hours. UPLC total run time is 10 min, including wash steps. Method performance: The limit of detection (LOD) for E2 and E1 was estimated to 0.3 pmol/L and 0.2 pmol/L, respectively. The limit of quantification (LOQ) was 0.6 pmol/L (E2) and 0,3 pmol/L (E1). Precision: the CV was below 9.0 % at all QC levels for E2, and 7.8 % for E1. The method is free of matrix-effects and is traceable to the E2 reference standard BCR576. Using healthy blood donors, reference ranges for E2 and E1 in postmenopausal women (>55 years, no estrogen supplements) were obtained. We confirmed ultra-low E2 and E1 levels in sera from patients using aromatase inhibitors such as Letrozole and Exemestane.

Conclusions: We have developed and validated a high-throughput LCMS/MS method for sub-picomolar levels of E2 and E1 and demonstrated its utility for postmenopausal breast cancer patients on AI. The assay is characterized by excellent accuracy and precision, and is traceable to a certified reference standard. The method was in February 2019 implemented in our lab for routine assessment and follow up of breast cancer patients.