An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of 17β-estradiol in human serum and plasma.

Objectives: A new candidate isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based reference measurement procedure (RMP) has been developed for the accurate and precise quantification of 17β-estradiol (E2) in human serum and plasma covering a measurement range from 0.400 to 5,000 pg/mL (1.47-18,357 pmol/L). To address this broad range, two separate methods were created: a high sensitivity (HS) method for concentrations between 0.400 and 5.00 pg/mL (1.47-18.4 pmol/L) and a standard range (SR) method for concentrations between 5.00 and 5,000 pg/mL (18.4-18,357 pmol/L).

Methods: As the primary reference material, E2 (CRM 6004-a) from the National Metrology Institute of Japan was used to ensure traceability to the international system (SI). A two-dimensional heart-cut LC approach was utilized for LC-MS/MS analysis, employing a supported liquid extraction sample preparation protocol for the SR method and a liquid-liquid extraction protocol followed by derivatization for the HS method. Assay validation was conducted following current guidelines. Selectivity and specificity were assessed using spiked serum samples, while potential matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision, accuracy, and trueness were determined using an extensive 5-day protocol. Standard measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM), with three individual sample preparations performed on at least two different days. Equivalence with higher-order RMPs was demonstrated through participation in the CDC Steroid Hormones Standardization (HoSt) program.

Results: The RMP enabled the quantification of E2 within the range of 0.400-5,000 pg/mL (1.47-18,357 pmol/L), demonstrating no interference from structurally related compounds and no evidence of matrix effects. The relative mean bias of the SR method ranged from -2.4 to 1.9 % across all levels, including secondary reference materials and spiked samples, whereas the HS method exhibited a mean bias ranging from -3.0 to 2.9 %. Expanded measurement uncertainties (k=2) for target value assignment ranged from 1.7 to 4.4 % for the SR method and were found to be ≤6.1 % for the HS method. The method's transferability was demonstrated in a comparison study at a second laboratory. Additionally, the candidate RMP exhibited excellent correlation and equivalence to JCTLM-listed RMPs through the CDC HoSt program.

Conclusions: In summary, the ID-LC-MS/MS-based RMP accurately quantifies E2. Its robust performance makes it suitable for standardizing routine assays and measuring individual patient samples, ensuring traceability.