Evaluation of an Isotope Dilution HPLC Tandem Mass Spectrometry Candidate Reference Measurement Procedure for Total 17-β Estradiol in Human Serum

Abstract

The inaccuracy of 17-β estradiol (E2) measurements affects its use as a biomarker in patient care and research. Clinical and research communities called for accurate and standardized E2 measurements. Reference Measurement Procedures (RMPs), part of the CDC Hormone Standardization Program (HoSt), are essential in addressing this need and ensuring that methods are accurate and comparable across testing systems, laboratories, and over time. A candidate RMP (cRMP) was developed for the measurement of total E2 in serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. The cRMP meets suggested performance criteria for accuracy and precision through the use of isotope dilution, calibrator bracketing, and gravimetric measurements. The cRMP demonstrated high agreement with certified reference materials (no significant bias to BCR576, 577, and 578) and established RMPs (slope 1.00, 95% CI 1.00–1.01; intercept 0.02, 95% CI -0.01 to 0.06). The cRMP is highly precise with intra-assay, interassay, and total percent CVs of 2.7%, 1.3%, and 2.4%, respectively. A higher specificity was achieved by measuring E2 without derivatization, compared to methods using derivatization agents. The cRMP can serve as a higher-order standard for establishing measurement traceability and provides an accuracy base against which routine methods can be compared in HoSt.

Quantitative determination of human serum testosterone via isotope dilution ultra‑performance liquid chromatography tandem mass spectrometry

“…ME. ME was evaluated in three matrices (charcoal-stripped serum, male serum and female serum) and one set of neat samples in methanol (matrix-free), according to previous studies (21,22). A 7-point calibration curve ranging from 10-1,000 ng/dl for testosterone was prepared in each matrix.…”

Simultaneous measurement of total estradiol and testosterone in human serum by isotope dilution liquid chromatography tandem mass spectrometry

“…Though a higher pH would increase the removal of these polar compounds (21), the pH of 8.0 was selected to be 2 pH units below the pKA of E2 (10.5). This avoids deprotonation and subsequent decrease in recovery of estradiol (29, 45). Repeating the entire two-step LLE procedure yielded in a higher extraction efficiency of 70% for E2 (95%CI: 67–72%) and 72% for TT (95%CI: 70–75%), compared to 43% for E2 (95%CI: 40–46%) and 44% for TT (95%CI: 43–46%) with only one extraction.…”

“…Agreement of the measured value with the assigned target value of the reference materials was assessed as described in NIST Special Publication 829 (24). In addition, measurement bias was evaluated following CLSI document EP9-A2 (25) using 40 single-donor serum materials with reference values assigned by higher-order reference methods listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database (NIST (26), University of Ghent (27, 28), and CDC Reference Laboratory (29, 30). The accuracy of the method was monitored over 2 years by measuring 10 blinded serum samples quarterly for each analyte with reference values assigned by higher-order reference methods.…”

“…Additionally, specificity was assessed by monitoring the QI/CI ratios in regular serum samples. A difference of more than ± 20% from the established target ratio was used to indicate the presence of an interfering compound (21, 23, 29, 34). …”

“…Sample matrix effects (ME) were assessed on four matrices (0.9% saline solution, synthetic serum, male serum, and female serum) and one set of neat samples in ethanol (matrix-free) using previously described procedures (29). An 8-point calibration curve ranging from 33.0–3670 pM (9–1000 pg/mL) for E2 and 0.31–34.7 nM (9–1000 ng/dL) for TT were prepared in each matrix.…”