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Evaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry

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dc.abstract.enWe evaluate here different analytical strategies for the chromatographic separation and determination of N-acetyl-5-methoxytryptamine (MEL) and its oxidative metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK) and cyclic 3-hydroxymelatonin (c3OHM) in cell culture samples. Two dimensional liquid chromatography (2D-LC) in the multiple heart-cutting mode was compared with regular 1D chromatographic separations of MEL and its oxidative metabolites. Our results showed that the use of trifluoroacetic acid (TFA) as mobile phase modifier was required to obtain a satisfactory resolution and peak shapes particularly for c3OHM. As TFA is not compatible with ESI ionization the application of the MHC mode was mandatory for a proper chromatographic separation. We evaluate also different internal standardization approaches based on the combined use of a surrogate standard (5-methoxytryptophol) and an internal standard (6-methoxytryptamine) for MEL quantification in cell culture samples obtaining unsatisfactory results both by 1D- and 2D-LC-ESI-MS/MS (from 9 ± 2 to 186 ± 38%). We demonstrate that only the application of isotope dilution Mass Spectrometry through the use of an in house synthesized 13C isotopically labelled analogue provided quantitative MEL recoveries both by using 1D- and 2D-LC-ESI-MS/MS (99±1 and 98±1. Respectively) in androgen-insensitive human prostate carcinoma PC3 cells.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorRodríguez-González, Pablo
dc.contributor.authorMenéndez, Rosa M. Sainz
dc.contributor.authorBarrallo, Juan Carlos Mayo
dc.contributor.authorCalleja, Javier García
dc.contributor.authorNaveda, Francisco Artime
dc.contributor.authorGago, Adriana González
dc.contributor.authorFernández, Amanda Suárez
dc.contributor.authorAlonso, J. Ignacio García
dc.contributor.authorZawadzka, Anna
dc.contributor.authorCzarnocki, Zbigniew
dc.date.accessioned2024-01-24T23:38:09Z
dc.date.available2024-01-24T23:38:09Z
dc.date.copyright2021-12-17
dc.date.issued2022
dc.description.accesstimeAT_PUBLICATION
dc.description.financePublikacja bezkosztowa
dc.description.versionFINAL_PUBLISHED
dc.description.volume1663
dc.identifier.doi10.1016/J.CHROMA.2021.462752
dc.identifier.issn0021-9673
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/106548
dc.identifier.weblinkhttp://dx.doi.org/10.1016/j.chroma.2021.462752
dc.languageeng
dc.pbn.affiliationchemical sciences
dc.relation.ispartofJournal of Chromatography A
dc.relation.pagesart.no. 462752
dc.rightsCC-BY-NC-ND
dc.sciencecloudnosend
dc.subject.enMelatonin
dc.subject.enMultiple heart cutting
dc.subject.enInternal standardization
dc.subject.enIsotope Dilution
dc.subject.enElectrospray
dc.titleEvaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry
dc.typeJournalArticle
dspace.entity.typePublication