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Authors: Chiara Calaprice, Caroline Le Goff, Etienne Cavalier
In clinical chemistry laboratories, the measurement of catecholamines in human plasma, in particular norepinephrine (NE), epinephrine (E), and dopamine (DP), is aimed to the diagnosis of neuroendocrine tumors, such as pheochromocytoma and neuroblastoma.
Many research and routine laboratories analyze this panel using extraction on alumina and electrochemical detection (HPLC-ECD). An isotope dilution method based on solid phase extraction (SPE) and reverse phase (RP) LC-MS/MS is under development. Catecholamines represent a challenge for RP LC-MS/MS for many reasons: first of all their polarity and their poor retention on RP classical C18 columns, as well as inadequate separation of normetanephrine and epinephrine which are isobaric compounds and might both be present in real samples. Also, their low level in plasma (low ng/L) is difficult to attain because of low recoveries and ion suppression from sample preparation.
Our LC-MS/MS system was a Nexera X2 UPLC from Shimadzu (Shimadzu Corporation, Kyoto, Japan) coupled to a QT5500 triple quadrupole mass spectrometer by Sciex (AB Sciex, CA, USA) fitted with an IonDrive™Turbo V.
- Retention of catecholamines on RP chromatographic columns:
Catecholamines are polar compounds. In order to promote interactions with stationary phase and have chromatographic resolution, very polar columns have been tested: Phenomenex Luna Omega polar C18 (1.6 µm, 100 × 2.1 mm) and Kinetex PFP column (100 Å, 1.7 µm, 2.1 mm × 100 mm). Eventually, PFP column was selected as general noise level was lower. Mobile phases were water and methanol, both 0.2% formic acid. Still to facilitate interactions with polar stationary phase, solvent gradient started at 0% organic phase, as even few µL of organic solvent lead to total co-elution. Post-run equilibration time was calculated to eliminate all traces of organic solvent from the column and keep the same resolution in consecutive runs.
- Sensitivity at ppt level:
High sensitivity was reached when using mobile phases with 0.2% formic acid instead of more common 0.1%.
– Sample preparation, SPE:
SPE with weak cation exchange resin was used. Different procedures and 96-well plates were tested. Very high ion suppression was remarked in some instances: elution with acidified organic solvent (MeOH 1% or 5% formic acid) resulted in low recoveries and significant ions suppression, also related to evaporation and solvent exchange. Eventually elution with water:iso-propanol 85:15, 0.1% formic acid allowed cleaned extracts and good recoveries, in the range of 60-120%, with no matrix effect. In this case, no solvent exchange was needed. Injection volume had to be 10µL max as the iso-propanol in the injection solvent caused loss of resolution.
A method for the detection and quantification of catecholamines in human plasma is under development and still needs to be tested for selectivity and sensitivity on real samples.
References & Acknowledgements:
IP Royalty: no
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