An Intact ACTH LC-MS/MS Assay as an Arbiter of Clinically Discordant Immunoassay Results

Clinical Chemistry
Junyan ShiMari L DeMarco

Abstract

Measurement of plasma adrenocorticotropic hormone (ACTH) is key in the differential diagnosis of hypothalamic-pituitary-adrenal disorders. Two-site sandwich immunoassays dominate clinical testing of ACTH in North America; however, discordant results between manufacturers have been repeatedly reported. To resolve the discrepancy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the intended measurand, biologically active intact ACTH (iACTH). The multiple reaction monitoring LC-MS/MS assay was designed to selectively measure full-length iACTH, as well as ACTH analogs and fragments (i.e., ACTH1-24 and ACTH18-39). Epitope assignment of the Roche Elecsys antibodies was performed by MALDI-TOF mass spectrometry. A method comparison between Roche Elecsys and Siemens Immulite ACTH immunoassays was performed and clinically concordant/discordant results identified. In a subset of these samples, the iACTH concentration was determined using the LC-MS/MS method. The lower limit of the measuring interval of the iACTH LC-MS/MS assay was 9 pg/mL (2 pmol/L). The assay was linear from 9 to 1938 pg/mL (2 to 427 pmol/L). Epitope mapping revealed that the Roche capture and detection antibodies bound residues 9-12 an...Continue Reading

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Citations

Jan 1, 2021·The Journal of Clinical Endocrinology and Metabolism·Jacquelien J HillebrandAnnemieke C Heijboer
Mar 27, 2021·Reviews in Endocrine & Metabolic Disorders·Zuleyha KaracaFahrettin Kelestimur
Sep 2, 2021·Journal of Neuroscience Methods·Taylor D PobranMari L DeMarco
Feb 8, 2022·Hormone and Metabolic Research = Hormon- Und Stoffwechselforschung = Hormones Et Métabolisme·Vikrant GosaviTushar Bandgar

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