Magdalena Rajska (Presenter)
Spadia Lab
Authorship: Magdalena Rajska (1), Petra Prochazkova (2), Denisa Bartonikova (3), Lenka Weiperova (4), Jakub Minar (5), Martin Radina (6)
Spadia Lab, Dr. Slabihoudka 6232/11, Ostrava-Poruba, 708 52
| Short Abstract Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, represent a clinically and etiologically diverse group of disorders. These tumors exhibits increased production of catecholamines and their metabolites and therefore quantification of biogenic amines is used for their diferentional diagnosis. The aim of this work was to develop the LC-MS/MS method based on hydrophilic interaction liquid chromatography which provides possibility for quantification of wider group of analytes related with neuroendocrine tumors. Method for quantification of metanephrines in urine fulfilled validation requirements. |
Long Abstract
Neuroendocrine tumors, such as pheochromocytomas or paragangliomas, represent a clinically and etiologically diverse group of disorders. These tumors exhibit a persistent or pyroxysmal excessive catecholamines production. Free metanephrines in plasma are prioritized for the diagnosis of pheochromocytoma due to their highest diagnostic sensitivity and specificity but in rutine practice for differential diagnosis of neuroendocrine tumors doctors often require parallel determination of urinary metabolites. The aim of these work was development and implementation of method for determination of metanephrines in urine by the liquid chromatography with tandem mass spectrometry (LC-MS/MS) suitable for routine practice in diagnostic laboratory which overcomes inconveniences related with classical liquid (HPLC) or gas chromatography (GC), like extensive sample preparation, the step of derivatization and long runtime.
Method development and validation were performed on Agilent 6490 Triple Quadrupole LC-MS/MS system. Chromatographic separation was achieved on SeQuant®ZIC®HILIC HPLC 3,5 µm, 100Å,PEEK 100 x 2,1 mm metal-free HPLC Column (Merc KGaA, Germany) by gradient elution. For determination of analytical parameters standards (purchased from Sigma Aldrich) and lyophilised controls (purchased from Recipe Chemicals and Instruments GmbH) were used.
The analytical performance of the developed method is satisfactory. The intra-assay coefficients of variation (CV) were below 5 %. Recoveries determined by method standard addition was from 91 to 101 %. Calibration range for metanephrine (MN) was 0,020 – 10,140 umol/l (R=0,999). Calibration range for normetanephrine (NMN) was 0,021 – 10,890 umol/l (R=0,999). The limit of quantification for MN was 0,020 umol/l, for NMN was 0,021 umol/l.
Presented method fulfilled validation requirements for both analytes and provides simple sample preparation, short runtime, high analytical sensitivity, specificity and accuracy. Presented configuration of the analytical column and mobile phases allows quantification of a wider group of analytes related with neuroendocrine tumors such as free plasma metanefrines and urinary metabolites of catecholamines - vanillylmandelic acid, homovanillic acid and 5-hydroxyindolacetic acid.
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