Anna Robson (Presenter)
Heart of England NHS Foundation Trust
Bio: I obtained my undergraduate degree in biological sciences (experimental pathology honours) at the university of Edinburgh, after which I moved to the University of Glasgow to undertake a research masters in proteomic technologies. I did my PhD at the University of Strathclyde where I used nanoparticle technologies and surface-enhanced resonance Raman scattering (SERRS) for the development of a novel immunoassay to screen for potential chemotherapeutic compounds. After completing my PhD I left academia and accepted a position as a Technical support specialist at Waters Corporation. I am currently a trainee clinical biochemist at the Heart of England NHS Foundation Trust.
Authorship: Anna Robson, Craig Webster
Heart of England NHS Foundation Trust, UK
| Short Abstract Analysis of both urinary free catecholamines and metanephrines are used in the diagnosis of phaeochromocytoma. This poster describes the development, validation and implementation of a new SPE-LC-MS/MS (solid phase extraction – liquid chromatography - tandem mass spectrometry) method for urinary free catecholamines and advantages of this method compared to the HPLC-ECD method previously used. The poster also demonstrates the capability of this new method to incorporate both catecholamines and metanephrines in a single analysis. |
Long Abstract
Measurement of both urinary catecholamines and metanephrines provides a specific and sensitive diagnosis of phaeochromocytoma. This poster describes the development of a novel SPE-LC-MS/MS (solid phase extraction – liquid chromatography - tandem mass spectrometry) method capable of quantifying urinary free catecholamines and metanephrines in a single analysis. Validation and implementation of this method is described for catecholamine analysis along with developments for the incorporation of metanephrines. Advantages of the new method, compared to the previously used HPLC-ECD (electrochemical detection), for routine use in a clinical laborartory are also demonstrated.
Catecholamines and metanephrines are extracted from urine via solid phase extraction (SPE) using WCX Evolute Express plates (Biotage). Analytes are eluted from SPE plates in aqueous solution (0.05% formic acid) and analysed by LC-MS/MS. Separation is achieved on a Kinetex F5 column using a gradient time program with 10mM ammonium formate (pH 3.5) and methanol. MS detection is attained using electrospray ionization and multiple reaction monitoring (API6500, Sciex).
Accuracy of the SPE-LC-MS/MS method was verified by running EQA samples and method comparison studies showed no significant differences to the HPLC-ECD method for noradrenaline and adrenaline. Dopamine results show a negative bias compared to HPLC-ECD, however this bias is not clinically significant and results are in comparable with other laboratories on the EQA scheme. Adrenaline results were found to be the most precise in imprecision studies, while dopamine results exhibited highest imprecision. The total allowable error was calculated as 18.4-19.0% (noradrenaline), 6.2-10.6% (adrenaline) and 15.7-23.4% (dopamine). The limits of quantification for routine analysis were defined as 40, 3 and 150 nmol/L for noradrenaline, adrenaline and dopamine respectively.
The SPE-LC-MS/MS method has numerous advantages over the previously used HPLC-ECD method. Specificity of the LC-MS/MS method mean it is subject to fewer interferences, thus allowing for accurate quantification of analytes in samples for which results could not be reported using HPLC-ECD. A reduced runtime allows for higher sample throughput and MRM permits quantification of catecholamines and metanephrines in a single run.
A novel SPE-LC-MS/MS method for urinary free catecholamine analysis has been successfully validated and implemented into a clinical laboratory for routine use. The method is shown to be capable of measuring both catecholamines and metanephrines, however quantification of metanephrines is not yet fully validated. This method also lends itself to in vivo and in vitro drug interference studies.
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