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2018-09-12 - Colloque/Présentation - communication orale - Anglais - page(s)

Lenaerts Charline , Wells Mathilde , Hambye Stéphanie , Blankert Bertrand , "Quantification of endogenous marinobufagin by a high-specific and very sensitive UPLC-MS/MS assay in non-patological pregnancy and preeclampsia" in DA-PBA 2018 : 11th International symposium on Drug Analysis - 29th International Symposium on Pharmaceutical and Biomedical Analysis, Leuven, Belgium, 2018

  • Codes CREF : Analyse et contrôle pharmaceutique (DI3450), Chimie analytique (DI1314), Sciences pharmaceutiques (DI3400)
  • Unités de recherche UMONS : Analyse pharmaceutique (M130)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé)
  • Centres UMONS : Centre de Recherche UMONS-Ambroise Paré (UMHAP), Centre Interdisciplinaire de Spectrométrie de Masse (CISMA)

Abstract(s) :

(Anglais) Marinobufagenin (MBG) is a bufadienolide cardiotonic steroid implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG has vasoconstrictive and cardiotonic properties by inhibiting the α1-isoform of the Na+,K+-ATPase pump, causing hypertension and natriuresis. Elevated endogenous MBG-like material has been described by poor-specific immunoassays in rat models of salt-sensitive preeclampsia as well as in preeclamptic human patients [1,2]. As it appears prior the development of the main symptoms of PE, MBG might be considered as one of the potential biomarkers for PE [3]. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence [4,5]. This led us to optimize a highly specific and sensitive analytical method relying on LC-MS/MS to measure MBG plasma levels. Pure MBG standard was obtained by extraction and purification from Bufo Marinus toad venom. Identity of the purified standard was confirmed using TLC-MS, QTOF and RMN. Thanks to pure MBG we have developed a high-specific and ultra-sensitive UPLC-MS/MS assay able to determine MBG in human plasma. Testosterone-d3 was used as internal standard and 2 mass transitions were recorded for each compound. A plasma extraction procedure based on SLE was elaborated to concentrate and clean the plasma sample prior to its analysis. The method is now being applied to a local clinical study allowing us to measure MBG plasma levels and biological parameters in preeclamptic women compared to non-pathological pregnancies. The developed LC-MS/MS assay was fully validated over a concentration range of 10 pg/mL to 500 pg/mL plasmatic MBG based on an internationally recognized validation strategy [6]. Accuracy profiles with one-sided 95% confidence interval and 30 % acceptance limits, as accepted by the AAPS guidelines [7], were built to evaluate the total error of the method. Matrix-effect and method recovery were evaluated for 4 different concentrations (n = 3) to 67.7% and 98.9% respectively. Stability tests, selectivity and QC analyses were also performed and will be presented. These results give us the opportunity to investigate MBG in non-pathological pregnancy as well as in preeclampsia. Preliminary results obtained by a previous LC-MS/MS assay allowed us to authenticate the presence of MBG in non-pregnant women as well as in early pregnancy. These preliminary pioneering results are giving a promising new perspective for preeclampsia risk assessment in pregnancy. References [1] Lopatin, D.A., et al., Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia. Journal of Hypertension, 1999. 17(8): p. 1179-1187. [2] Agunanne, E., et al., Marinobufagenin Levels in Preeclamptic Patients: A Preliminary Report. American Journal of Perinatology, 2011. 28(7): p. 509-514. [3] Vu, H.V., et al., Involvement of Marinobufagenin in a Rat Model of Human Preeclampsia. American Journal of Nephrology, 2005. 25(5): p. 520-528. [4] Abi-Ghanem, D., et al., A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids. Journal of Immunoassay and Immunochemistry, 2011. 32(1): p. 31-46. [5] Fedorova, O.V., et al., Endogenous Ligand of α1 Sodium Pump, Marinobufagenin, Is a Novel Mediator of Sodium Chloride–Dependent Hypertension. Circulation, 2002. 105(9): p. 1122-1127. [6] Hubert, P., et al., Harmonization of strategies for the validation of quantitative analytical procedures: A SFSTP proposal – Part II. Journal of Pharmaceutical and Biomedical Analysis, 2007. 45(1): p. 70-81. [7] Viswanathan, C.T., et al., Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays. Pharmaceutical Research, 2007. 24(10): p. 1962-1973.