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

The inaccuracy of 17-ß 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.

Practical considerations for accurate determination of free thyroxine by equilibrium dialysis.

Background: Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the "gold standard" for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration. Methods: In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions. Results: The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze-thaw cycles, 5 °C for 18-20 h, or -70 °C for a minimum of three years. Conclusion: The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.