Development of an Immunoprecipitation Method for the Analysis of Intact Parathyroid Hormone (PTH) and Related Forms by LC-MS/MS
Jordi Farré-Segura, Justine Demeuse, Caroline Le Goff, Etienne Cavalier Department of Clinical Chemistry, University Hospital of Liège, University of Liège, Belgium.
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Jordi Farré-Segura (Presenter) University of Liège
Presenter Bio: Jordi Farré obtained his degree and master-equivalent diploma in pharmacy in September 2017 by University of Barcelona. In 2016, he was firstly introduced to the clinical chemistry field in his hometown (Manresa) hospital’s laboratory as a trainee. Later this same year he was granted with an Erasmus exchange to Liège, where he develops his research in bone biomarkers since October 2017 as a PhD student at the Department of Clinical Chemistry, University of Liège (Belgium) under the supervision of Prof. Etienne Cavalier and Dr. Anne-Catherine Servais.
He was granted a Young Investigator Grant for MSACL EU 2018 where he did an oral communication entitled “A newly developed LC-MS/MS method for the quantitation of Glucagon in plasma shows the lack of specificity of a former immunoassay method”.
Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
INTRODUCTION:
Parathyroid hormone measurement has faced cross-reactivity issues ever since the first assays were developed. Multiple hormone fragments found in the body hamper the specific measurement of clinically relevant forms when using immunoassays. Therefore, the use of LC-MS/MS techniques could provide the required specificity for unequivocal PTH analysis as has recently been demonstrated by some published methods, which rely on immunoprecipitation of the analyte followed by a tryptic digestion step.
Considering the length of the whole PTH (84 amino acids) and its molecular weight of 9425 Da, an intact protein analysis seems a practicable approach as described in the present project.
OBJECTIVES:
The aim of this work was to develop a method to quantify intact PTH hormone, including its oxidized forms by LC-MS/MS, capturing the compound with antibody coated magnetic beads. Other PTH fragments are also in the scope for further project development.
METHODS:
Proposed sample preparation
Sample preparation was performed by means of immunoprecipitation by magnetic beads coated with protein G. Beads were initially washed three times in a 1.5 mL vial with a 0.1% solution of PBS-Tween 20 (Washing solution), then 200 µL of a 5 µg/mL solution of monoclonal antibodies (targeting C-terminal PTH) was added and incubated together with the beads for 10 min at room temperature prior to a three times wash. 500 µL of sample was added to the beads, incubated for 1h at room temperature and washed once. Finally, the captured analyte was eluted with a solution containing 50% acetonitrile and 0.5% formic acid. The eluate was evaporated in a SpeedVac, reconstituted in 10% acetonitrile and 0.01% formic acid and further injected to a Nexera X2 ultraperformance liquid chromatography (UHPLC) coupled to a Sciex QTRAP® 6500 triple quadrupole MS/MS in positive electrospray mode.
RESULTS:
Preliminary results
Plasma free samples spiked with intact PTH to a concentration of 500 pg/mL gave intense peaks in the range of 105 counts per second with a very low background noise. Signal-to-noise ratio (S/N) for these samples was calculated 600. Other forms of PTH such as 7-84 and 1-84 oxidized forms were also isolated and successfully analyzed by means of this technique.
CONCLUSION:
This innovative intact immunoprecipitation LC-MS/MS approach could provide the required selectivity to unambiguously quantitate PTH and some of its truncated forms. The good response found in the preliminary results suggest the suitability of the method for low concentrated samples, as they are found in the body.
= Discovery stage.
= Translation stage.
= Clinically available.
