MSACL 2016 EU Abstract

Simultaneous Extraction of Catecholamine and Metanephrines from Plasma Prior to Analysis Using LC-MS/MS

Alan Edgington (Presenter)
Biotage (GB) Ltd

Bio: Senior Scientist at Biotage (GB) Ltd. Responsible for supporting the development of application notes for sample preparation and the Extrahera.

Authorship: Alan Edgington 1
1 Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK

Short Abstract

This poster discusses the impact of optimization of various parts of the method development process to maximise sensitivity while delivering a combined assay for the analysis of plasma catecholamines and metanephrines. Method parameters; precursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimized for increased sensitivity. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC system coupled to an AB SCIEX 5500 triple quadrupole MS. SPE methods demonstrated recoveries greater than 80 % with RSDs below 10 %. Calibration curves from 20 to 1280 pg/mL demonstrated good linearity and r2 values greater than 0.99 for all analytes.

Long Abstract

Introduction

Catecholamines and metanephrines continue to be important biomarkers related to various neurological issues. This poster discusses the impact of optimization of various parts of the method development process to maximise sensitivity and ultimately result in a combined assay. Method parameters; precursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimized for increased sensitivity.

Methodology

LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC system coupled to an AB SCIEX 5500 triple quadrupole MS. Optimized MRM transitions were selected using the most intense precursor ions. LC mobile phases and stationary phase were selected based on analyte retention, resolution, symmetry and MS signal to noise. Catecholamine, metanephrines and their internal standards were spiked into human plasma at various concentrations. Sample preparation utilized polymer-based mixed-mode weak cation exchange SPE. Strategies focused on sample pre-treatment, loading volumes, extraction pH, wash solvent combinations for extract cleanliness, elution solvent combination and minimum elution volumes.

Results

MS optimization demonstrated increased sensitivity and specificity using in-source fragmentation to form dehydrated precursor ions, [M H2O+H]+ for epinephrine, norepinephrine, metanephrine and normetaneprine. The same effect was not demonstrated for dopamine or 3-methoxy-tyramine due to the absence of an alpha hydroxyl moiety on the side chain. Analyte volatility resulted in substantial evaporative losses for the catecholamines. Acidification or the addition of ethylene glycol did not prove successful in this case.

SPE optimization was tailored for the extraction of the catecholamines. Due to the presence of the cis-diol group on the ring system these were a far more challenging analyte panel compared to the metanephrines. The optimized SPE method required low ionic strength and pH control around 7 ensuring analytes and sorbent are in their ionized forms. Optimized wash protocols demonstrated no interference from phospholipids. Minimum elution solvent volumes were performed using a 10 mg EVOLUTEĀ® EXPRESS WCX plate. The combination of low volume and high aqueous proportion of the elution solvent allowed the elimination of the evaporation step resulting in reduced variability. Due to the presence of water wettable components it was also possible to eliminate plate conditioning steps prior to sample loading. SPE methods demonstrated recoveries greater than 80 % with RSDs less than 10 %. Calibration curves constructed using spiked plasma from 20 to 1280 pg/mL demonstrated good linearity and coefficients of determination greater than 0.99 for all analytes. Signal to noise ratios at 20 pg/mL were demonstrated to be much greater than 10:1.

Conclusion

This poster demonstrates the simultaneous extraction of catecholamines and metanephrines from human plasma. The weak cation exchange SPE protocol provided sufficient extraction recovery and extract cleanliness in order to reach limits of quantitation below 20 ng/mL for both analyte panels.


References & Acknowledgements:

Full list of Authors

Alan Edgington 1, Adam Senior 1, Lee Williams 1, Rhys Jones 1, Helen Lodder 1, Geoff Davies 1, Steve Jordan 1, Claire Desbrow 1, Paul Roberts 1, Stephanie Marin 2, Dan Menasco 2 & Elena Gairloch 2.

1 Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK.

2 Biotage, 10430 Harris Oaks Blvd., Suite C, Charlotte, North Carolina 28269, USA.


Financial Disclosure

DescriptionY/NSource
Grantsno
SalaryyesBiotage (GB) Ltd
Board Memberno
Stockno
ExpensesyesBiotage (GB) Ltd

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

yes