Development and validation of a candidate reference method for 1-84 PTH by LC-MS/MS

BACKGROUND-AIM The accurate measurement of parathyroid hormone (PTH) concentrations is of paramount importance, especially for patients with parathyroid gland disorders and for monitoring bone turnover in individuals with chronic kidney disease and mineral bone disorders. Two generations of PTH immunoassays (IA) are currently present on the market, presenting known cross-reactivity issues and a lack of standardization. Some higher-order techniques, such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), are currently being explored as potential newest generation assays featuring unprecedented specificity and sensitivity. We have developed and validated the first LC-MS/MS method with antibody-free sample preparation, aiming to become a candidate reference measurement procedure for 1-84 PTH. METHODS Separation and quantification of full length 1-84 PTH were achieved by a Shimadzu Nexera X2 UPLC coupled to a Sciex QT6500 MS. LC was performed on an Acquity UPLC BEH C4 300Å column using a mobile phase gradient made of water and -acetonitrile containing 0.4% formic acid and 5% Dimethylsulfoxide. MS multiple reaction monitoring recorded 3 transitions for PTH and its isotopically labeled internal standard for specificity purposes. Sample preparation was done on patients’ serum or EDTA-K2 plasma through a solid-phase extraction. The method was validated following the Clinical and Laboratory Standards Institute C-62A guidelines. Forty-four external quality controls (EQC) from UK NEQAS were used to verify LC-MS performance. Patient samples analyzed by LC-MS/MS and Diasorin Liaison 3rd generation IA were compared. RESULTS Validation was successfully completed, with a lower limit of quantification of 5.0 pg/mL. Analytical CV ranged from 6 to 11% with measurement uncertainties of 8 to 12% along the measuring range 5-1000 pg/mL. Good agreement wasobtained between LC-MS/MS and IA in patients and EQC. CONCLUSIONS We present the first fully validated LC-MS/MS method for the quantitation of 1-84 PTH with antibody-free samplepreparation. The excellent analytical performance allow us to present this method as a candidate referencemeasurement procedure