LAUSR

Abstract With a rising incidence in renal failure, chronic kidney disease (CKD) has become one of the leading health conditions in the U.S. The earlier stages of CKD generate few symptoms and only until the kidney is significantly impaired do patients begin experiencing signs of renal failure. Parathyroid hormone (PTH) is a key biomarker in patient diagnosis and treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD), as well as hypo/hyperparathyrioidism. Elevated levels of intact PTH (iPTH) have been associated with CKD Stages 3-5. The most recent update of the Kidney Disease Improving Global Outcomes (KDIGO) guideline for CKD-MBD management emphasized the role of PTH as one of the key biomarkers of this disorder. Intact PTH, secreted normally into the blood at 10-65 pg/mL concentration levels, can undergo oxidative or enzymatic metabolic cleavages within parathyroid glands or peripheral tissues to produce N-terminal, C-terminal, and mid-range fragments. The ratio of these PTH fragments to iPTH has been diagnostic for severe or end-stage renal disease, non-dynamic bone disease, and hyperparathyroid-associated bone loss. Current PTH immunoassays demonstrate significant variability with up to a 4.2 fold difference in measurements depending on the method used. This variability can lead to misclassification of patients. Such significant variability has been attributed to differences in antibody specificity, lack of accurate calibration, matrix effects, or interferences. This creates the need for a new accurate and specific method to measure PTH and its fragments. In addition, the low endogenous concentration of PTH in biological matrices requires both, a highly sensitive and specific methodology. The CDC Clinical Standardization Programs in collaboration with the Partnership for the Accurate Testing of Hormones is addressing this need. This work highlights a novel high resolution mass spectrometry-based reference measurement procedure for intact PTH (1-84) and its fragments. Mass spectrometric immunoassay (MSIA) pipette tips were embedded with antibodies for PTH immunocapture. Analytes were eluted from the MSIA tips and analyzed directly with reversed phase ultra-high liquid chromatography high-resolution mass spectrometry (RP-UHPLC-HRMS). Initial results demonstrate the ability to quantify intact PTH and PTH fragments with high specificity. Calibration curves using stable isotope labeled internal standards were generated for iPTH, N-terminal PTH fragments, C-terminal PTH fragments, and mid-region PTH fragments. Initial assessments demonstrate excellent linearity (R2 = 0.991-0.997), reproducibility, and sufficient sensitivity of the UHPLC-HRMS system with a linear range of 15-3000 pg/mL PTH. This method was applied to samples from healthy patients and patients with different CKD stages. PTH and its fragments were detectable in these samples.