A-155 Establishing a Liquid Chromatography Mass Spectrometry Reference Measurement Procedure for the Accurate Quantification of Parathyroid Hormone 1-84

Abstract

Background Parathyroid hormone (PTH) is a widely accepted diagnostic biomarker for Chronic Kidney Disease-Mineral and Bone Disorder (CKD), as well as hypo- and hyperparathyroidism.[1] Current clinical immunoassays measuring PTH(1-84) yield variable results, which limits the clinical utility of PTH as a diagnostic biomarker and can lead to patient misclassification, underscoring the need for standardization to improve assay measurement agreement.[2, 3] The Centers for Disease Control is developing a novel liquid chromatography mass spectrometry (LC-MS) reference measurement procedure (RMP) to accurately quantify PTH(1-84) in human specimens. Methods A LC-MS method was developed to accurately quantify PTH (1-84). To effectively isolate and enrich PTH(1-84) from human specimens, an immunoprecipitation (IP) method that uses magnetic beads coated with N- and C-terminal epitope antibodies was established. Beads are added to samples, mixed for 1 hour, washed 4 times, and then PTH is eluted for analysis by LC-MS. LC separations are conducted at a capillary flow rate of 3 µL/min and reverse phase trap-elute and analytical reverse-phase chromatography approaches are employed. MS measurements use multiple reaction monitoring with a triple quadrupole instrument. Endogenous PTH(1-84) is quantified relative to a stable isotope labeled (SIL) internal standard. Results A calibration curve spanning a concentration range of 0.74-2,200 pg/mL was established for the PTH RMP. The IP method was optimized to capture PTH(1-84), and the corresponding SIL, with 99.5% average recovery. Linear regression analysis yielded R² values of = 0.99. The estimated limit of detection for the PTH(1-84) LC-MS method is 0.74–1.84 pg/mL with 3–8% average relative error in method serum calibrators. The PTH RMP was applied to 125 commercially sourced plasma samples, all of which were analyzed in duplicate. PTH concentrations in plasma ranged from 0.39–81.73 pg/mL, which are lower than concentrations typically measured by clinical immunoassays, suggesting the PTH LC-MS RMP is more selective. Conclusion This novel, sensitive, and selective PTH LC-MS RMP allows for effective enrichment and accurate quantification of PTH(1-84). The PTH RMP will serve as the basis of the CDC’s PTH Clinical Standardization Program and will be used to value assign reference materials used for PTH standardization, ultimately improving the analytical accuracy and reliability of PTH assays used in patient care.