MSACL 2019 EU Abstract

Separation of Intact Parathyroid Hormone and Variants Using a Highly Sensitive Sheathless CE-ESI-MS/MS Method

Laurent Nyssen (1, 2), Marianne Fillet (2), Etienne Cavalier (1), Anne-Catherine Servais (2)
(1) Department of Clinical Chemistry, Center for Interdisciplinary Research on Medicines (CIRM), ULiege, CHU Sart-Tilman, (2) Laboratory for the Analysis of Medicines (LAM), Center for Interdisciplinary Research on Medicines (CIRM), ULiege

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Laurent Nyssen (Presenter) University of Liège >> POSTER (PDF)

Presenter Bio: Laurent Nyssen is a PhD student at the University of Liège and member of the Center for Interdisciplinary Research on Medicines. As a pharmacist, his interest lies in intact protein analysis using separative techniques, for both clinical and quality control purposes.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Moreover, immunoassays may cross-react with PTH variants such as C-terminal fragments stemming from PTH catabolism. These issues make it difficult to compare results obtained in different laboratories. A reference quantification method is necessary to harmonize PTH assays, both sensitive and selective enough to detect PTH at low concentrations among a variety of closely related compounds.
OBJECTIVES In this study, our main goal was to reach a very high sensitivity (pg/mL range) for the analysis of PTH and its variants. Two variants were selected, namely 7-84 PTH as C-terminal fragment and 1-34 PTH as related peptide, but also as potential internal standard for future works.
METHODS To achieve our goal, we developed a sheathless CE-ESI-MS method for the separation of 1-34 PTH, 7-84 PTH, and 1-84 PTH. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis (t-ITP), field-amplified sample injection (FASI) and electrokinetic supercharging (EKS).
RESULTS The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. 1-84 PTH (full length), 7-84 PTH and 1-34 PTH were separated using an acidic background electrolyte containing acetonitrile to reduce peptide adsorption onto the capillary wall. Ammonium acetate was used as sample medium to improve sensitivity through t-ITP. The method was then transferred to a sheathless CE-ESI-MS instrument. CE-MS on fused silica capillary was limited to µg/mL levels. Indeed, despite the MS detection, only samples containing at least 10 µg/mL of 1-84 PTH, 7-84 PTH, and 1-34 PTH could be analyzed. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH and 1-34 PTH were detected at 100 ng/mL using FASI while 1-84 PTH and 1-34 PTH were detected at 100 pg/mL using EKS. The estimated LODs (S/N = 3) for the EKS method were 25 pg/mL for 1-84 PTH and 10 pg/mL for 1-34 PTH, while there was no signal anymore for 7-84 PTH at these levels.
CONCLUSION The developed sheathless CE-ESI-MS method has the potential to reach the low pg/mL range in biological samples after the optimization of the sample preparation method.