Magdalena Rajska (Presenter)
SPADIA LAB a.s.
Authorship: Magdalena Rajska, Petra Prochazkova, Denisa Bartonikova, Lenka Weiperova, Veronika Plunderova, Peter Loucka, Minar Jakub, Martin Radina
Spadia lab, a.s., Dr. Slabihoudka 6232/11, Ostrava-Poruba, 708 52
| Short Abstract Neuroendocrine tumors, an uncommon group of cancers, represent clinically and etiologically diverse group of disorders. These tumors exhibit secretion of many hormonally active substances which cause various endocrine syndroms. The analysis of catecholamine metabolites, both in plasma and in urine, are of importance for the diagnosis of tumors such as pheochromocytomas or paragangliomas. In case of carcinoid the diagnostic assessment is based on determination of 5-HIAA in urine and serotonin in serum. The aim of this work was to develop methods in such a way to maintain the same configuration of the LC-MS/MS system. Methods for wider group of analytes were developed, as follows: metanephrine and normetanephrine in plasma and in urine; serotonin in serum; vanilmandellic, homovanilic and 5-hydroxyacetic acid in urine. Methods fulfilled validation requirements. |
Long Abstract
Neuroendocrine tumors (NETs), an uncommon group of cancers, represent clinically and etiologically diverse group of disorders. These tumors exhibit secretion of many hormonally active substances and thus cause various endocrine syndroms. Diagnostic alghorithm is based on the clinical symptomathology, morphological methods and laboratory diagnostics, for localization of NETs RTG methods are used. For the detection of NETs in early stage the estimation of hormonally active substances and their metabolites is very important. The analysis of catecholamine metabolites, both in plasma and in 24 hour urine collection, is of importance for the diagnosis of tumors of the sympathoadrenal system, such as pheochromocytomas or paragangliomas. In case of carcinoid tumors the diagnostic assessment is based on determination of 5-hydroxy-indolacetic acid in 24 hour urine collection and supported by additional determination of serotonin in serum.
The aim of this work was to develop methods in such a way as to maintain the same configuration of the LC-MS/MS system (instrument, analytical column and mobile phases). Methods for wider group of analytes relevant for diagnostic assessment of neuroendocrine tumors were developed for analytes as follows: metanephrine (MN) and normetanephrine (NMN) in plasma and in 24 hour urine collection; serotonine (SER) in serum; vanilmandellic (VMA), homovanillic (HVA) and 5-hydroxyindolacetic acid (5-HIAA) in 24 hour urine collection.
All above mentioned LC-MS/MS methods were based on hydrophilic interaction liquid chromatography (HILIC) and ESI+ or ESI- ionization. Sample preparation is simple, without the step of solid phase extraction and without the need of derivatization. Methods validation as well as determination of plasma/serum/urine concentration were performed on Agilent 6490 Triple Quadrupole LC/MS System. Chromatographic separation was achieved on SeQuant®ZIC®HILIC HPLC 3,5 µm, 100Å, PEEK coated 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 calibrators and controls (purchased from Recipe Chemicals and Instruments GmbH) were used.
Methods fulfilled validation requirements for all analytes. These methods are reliable in terms of precision. Intraassay and interassay precision values were below 10%. Recoveries at concentration within normal and at pathological concentration level ranged between ± 10%. The calibration curves provides linearity in the relevant ranges of concentration. The limits of quantification (LLQ, CVs below 15%) for MN in plasma was 56,6 pmol/l for NMN in plasma was 102,5 pmol/l, for MN in urine was 0,020 umol/l for NMN in urine was 0,021 umol/l, for SER in serum was 4,70 nmol/l, for VMA in urine was 0,786 umol/l, for HVA in urine was 0,855 umol/l and for 5-HIAA was 0,814 umol/l.
The advantages of presented LC-MS/MS methods over classical HPLC and immunoassay approaches include simple step of sample preparation, short runtime, high analytical sensitivity, specificity and accuracy. This methods are reliable in terms of precision and provides linearity in the relevant ranges of concentrations. This arrangement, one configuration of the LC-MS/MS system - instrument, analytical column and mobile phases, allows for easy changing between methods without the need of manual intervention.
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