A cardiotonic steroid multiplex method using ultra-high-performance liquid chromatography-tandem mass spectrometry
Authors: Gilligan Lorna C , Alamrani Daniyah , Fobian Dannie , Kotecha Dipak , Kirchhof Paulus , Arlt Wiebke , Taylor Angela E , Pavlovic Davor 
Published: October 2023

Background and Aims

Digoxin, a cardiotonic steroid (CTS), is commonly prescribed for patients with atrial fibrillation and heart failure. Endogenous CTS have been implicated in cardiovascular disease pathogenesis and can interact with digoxin. We developed an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of eleven CTS.

Materials and Methods

Isotopically labelled internal standards were added to samples, followed by protein precipitation and solid-phase extraction. Steroids were separated using an Acquity uPLC chromatography system with a Waters CORTECS T3 column (1.6 μm 2.1x50 mm) and quantification performed on a Waters TQ-XS mass spectrometer using electrospray ionisation in positive ion mode. Separation used a methanol/ water elution system containing 0.1% formic acid and post-column infusion of lithium chloride.

Results

Run time was 13.5 minutes. Lower limits of quantification ranged from 0.025 to 0.1 ng/ml. Serum recovery ranged from 40.0-98.6% with matrix effects from -22.9% to 7.6%. Plasma recovery ranged from 27.4-83.9% and matrix effects were -27.6-39.1%. Accuracy and precision at three concentrations were within ideal range (<15%) for seven CTS and <20% for the others.

Conclusion

This validated UHPLC-MS/MS method provides a comprehensive assessment profiling 11 cardiotonic steroids, offering a sensitive and specific tool for clinical and pre-clinical investigations.

Highlights

Mass spectrometry method simultaneously quantifies multiple cardiotonic steroids

Accurate and specific measurement of clinically relevant digoxin concentration

Method validated for measurement of cardiotonic steroids in serum and plasma

Post-column infusion of lithium chloride substantially improves sensitivity

Research funding

This work was funded by the British Heart Foundation (PG/17/55/33087, FS/PhD/22/29309, FS/19/12/34204, RG/17/15/33106 to DP, Accelerator Award AA/18/2/34218 to Institute of Cardiovascular Sciences), Wellcome Trust (Seed Award Grant 109604/Z/15/Z to DP) and Department of Clinical Laboratory Sciences, Faculty of Applied medical Sciences, University of Hail. PK was partially supported by European Union AFFECT-AF (grant agreement 847770), and MAESTRIA (grant agreement 965286), British Heart Foundation (PG/17/30/32961; PG/20/22/35093; AA/18/2/34218), German Centre for Cardiovascular Research supported by the German Ministry of Education and Research (DZHK), Deutsche Forschungsgemeinschaft (Ki 509167694), and Leducq Foundation. The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

Financial disclosures

Prof. Kotecha reports grants from the National Institute for Health Research (NIHR CDF-2015-08-074 RATE-AF; NIHR130280 DaRe2THINK; NIHR132974 D2T-NeuroVascular; NIHR203326 Biomedical Research Centre), the British Heart Foundation (PG/17/55/33087, AA/18/2/34218 and FS/CDRF/21/21032), the EU/EFPIA Innovative Medicines Initiative (BigData@Heart 116074), EU Horizon (HYPERMARKER 101095480), UK National Health Service -Data for R&D-Subnational Secure Data Environment programme, UK Dept. for Business, Energy & Industrial Strategy Regulators Pioneer Fund, the Cook & Wolstenholme Charitable Trust, and the European Society of Cardiology supported by educational grants from Boehringer Ingelheim/BMS-Pfizer Alliance/Bayer/Daiichi Sankyo/Boston Scientific, the NIHR/University of Oxford Biomedical Research Centre and British Heart Foundation/University of Birmingham Accelerator Award (STEEER-AF). In addition, he has received research grants and advisory board fees from Bayer, Amomed and Protherics Medicines Development; all outside the submitted work.

PK received research support for basic, translational, and clinical research projects from European Union, British Heart Foundation, Leducq Foundation, Medical Research Council (UK), the Deutsche Forschungsgemeinschaft (DFG) and German Centre for Cardiovascular Research, from several drug and device companies active in atrial fibrillation, and has received honoraria from several such companies in the past, but not in the last three years. PK is listed as inventor on two issued patents held by University of Hamburg (Atrial Fibrillation Therapy WO 2015140571, Markers for Atrial Fibrillation WO 2016012783).

Graphical Abstract