Lee Williams (Presenter)
Biotage GB Limited
Authorship: Alan Edgington1, Adam Senior1, Lee Williams1, Rhys Jones1, Helen Lodder1, Geoff Davies1, Steve Jordan1, Claire Desbrow1, Paul Roberts1, Victor Vandell2 & Elena Gairloch2.
1Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK.
| Short Abstract Catecholamines are classic biomarkers for the detection of diseases like hypertension, pheochromocytoma and neuroblastoma. The main target analytes - epinephrine, norepinephrine and dopamine, are traditionally analyzed using liquid chromatography with electrochemical detection. This poster discusses the impact of optimization of various parts of the method development process to maximise the sensitivity of LC-MS/MS analysis. A highly sensitive LC/MS system, a Shimadzu Nexera UHPLC coupled to an AB SCIEX 5500 triple quadrupole MS was used for analysis. Method parameters; pre-cursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimised for increased sensitivity, allowing quantitation down to 20 pg/mL. |
Long Abstract
Introduction
Catecholamines are biomarkers for the detection of diseases like hypertension, pheochromocytoma and neuroblastoma. The main target analytes -epinephrine, norepinephrine and dopamine, are traditionally analyzed using liquid chromatography with electrochemical detection. This poster discusses the impact of optimization of various parts of the method development process to maximise the sensitivity of LC-MS/MS analysis. Method parameters; precursor ion selection, MRM transitions, chromatography and solid phase extraction protocols were optimised for increased sensitivity.
Methodology
A Shimadzu Nexera UHPLC coupled to an AB SCIEX Triple Quad 5500 MS was used for analysis. Catecholamine standards were infused using a Turbo-V API source operating in positive ESI mode. Source parameters were optimized to enhance the production of precursor ions to increase signal sensitivity for epinephrine and norepinephrine. MRM transitions to product ions were selected from the most intense precursors. LC method development was performed with the aid of a column switching valve installed in the Shimadzu CTO-20 column oven allowing the comparison of 4 HPLC or UHPLC columns. The final column/eluent combination was chosen on the basis of analyte retention, resolution, symmetry and MS signal to noise. Catecholamines and their internal standards were spiked into human plasma at various concentrations. Sample preparation utilized polymer-based mixed-mode weak cation exchange SPE, specifically EVOLUTE® EXPRESS WCX in the 10 and 30 mg fixed well plate formats.
Results
MS optimization demonstrated in-source fragmentation to provide increased sensitivity and specificity of MRM transitions for dehydrated epinephrine and norepinephrine [M H2O+H]+ by a factor of at least 2. The same effect was not demonstrated by dopamine due to the absence of an alpha hydroxyl moiety on the side chain. Evaluation of UHPLC columns resulted in selection of an ACE C18-PFP chemistry due to superior retention, separation and peak shape. Final mobile phase selection based on chromatographic peak shape and maximum signal to noise was ammonium formate with a small addition of formic acid in water and methanol.
Due to the small polar nature of the analytes volatility issues during evaporation was observed in the form of analyte losses and signal variability. Attempts to reduce or eliminate these evaporative losses and variability using acidification or the addition of ethylene glycol did not prove successful.
SPE optimization investigated: sample pretreatment, maximum matrix volumes, pH control throughout the procedure, wash solvent combinations for extract cleanliness, elution solvent combination and minimum elution volumes. The optimized method required pH control with low ionic strength at a pH of around 7 ensuring that both analytes and the sorbent are in their ionized forms. Optimized wash protocols demonstrated no interference from phospholipids when extracting 250 µL of plasma. In order to minimize elution solvent volumes final SPE protocols were performed using a 10 mg EVOLUTE® EXPRESS WCX plate. The combination of low volume and large acidic aqueous proportion therefore allowed the elimination of the evaporation step. The final method demonstrated recoveries greater than 80 % with RSDs less than 10 %. Due to the presence of water wettable components it was possible to eliminate the plate conditioning steps. The load-wash-elute protocol resulted in recoveries great than 70% and RSDs below 10%. Calibration curves for both protocols were constructed using spiked plasma. Good linearity was observed from 20 to 1280 pg/mL, demonstrating 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 applicability of polymer-based mixed-mode weak cation exchange SPE for the extraction of catecholamines from plasma when combined with carefully optimized LC-MS/MS conditions to enhance analyte sensitivity. This resulted in a simple sample preparation workflow with no requirement for analyte derivatization and an extremely sensitive assay with LLOQs less than 20 pg/mL.
References & Acknowledgements:
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