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Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis.

Introduction Screening for pre-eclampsia is a focus of prenatal care and is largely based on the use of clinical risk factors. However, current screening protocols are unfit to determine pre-eclampsia risk in 1st time pregnant women; biomarkers have the potential to address this unmet need. As single biomarkers have insufficient predictive accuracy, unbiased biomarker discoveries have been performed to identify panels of markers which when combined, have the potential pre-eclampsia prediction. Metabolite based solutions using mass spectrometry have gained significant interest as they have the potential to readily translate to clinical practice. Objectives To translate the discovery that blood-borne metabolite biomarkers stratify pregnant women early in pregnancy (∼15 weeks) to their risk of pre-eclampsia [1] into a commercial LC–MS based clinical assay. To pursue fit-for-purpose testing of the first version of the developed LC–MS pipeline followed by independent verification through a case:control study. Methods The analysis pipeline incorporated (1) a single step metabolite extraction, (2) multiplex LC-QqQ-MS assays for 40+ metabolites and (3) a dedicated data processing protocol. Case:control study testing utilised 15 weeks’ plasma samples of from 50 pregnant women who subsequently developed pre-eclampsia and 500 random control pregnancies. All participants are part of the SCOPE study [2] and were recruited in Cork, Ireland. Results For the 40+ metabolite assays: 62% had a %CV £ 15% and 82% had a %CV £ 25%. Univariate analyses using the ROC statistic showed that 7 of the metabolites tested had significant predictive power (lower limit 95% CI ROC-AUC [3] 0.5). Multivariate logistic regression analysis revealed particular combinations of metabolites which identified groups of women either at increased risk or at decreased risk for pre-eclampsia. Conclusion These findings underpin the potential of metabolite-centric multimarker panels to encapsulate a complex syndrome such as pre-eclampsia considerably in advance of any clinical manifestation. Further development steps will include performing additional case:control studies and subsequent clinical validation of this metabolite based test in the large scale European, multicentre phase IIa clinical study IMproved Pregnancy Outcomes by Early Detection (IMPROvED) which is currently recruiting 1st time pregnant women in 5 European countries [3] . Acknowledgment The authors gratefully acknowledge funding from the EU-HEALTH Project IMPROvED (305169) of the Seventh Framework Programme (FP7).

Introduction Marinobufagenin (MBG) is an endogenous bufadienolide cardiac inotrope which is demonstrating growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE) and end-stage renal disease hypertension. Mammalian MBG is an inhibitor of the α 1 isoform of Na + , K + -ATPase with vasoconstrictive and cardiotonic properties, resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1,2,3]. The rise of endogenous MBG appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential target in the biomarker panel for PE. A sensitive and accurate analytical method is needed to assess MBG in as lower level as possible in plasma. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Objective Our aim is to develop a MBG assay using a more specific and easy to access technique, such as LC–MS/MS. An algorithm dealing with the MBG plasma levels might be established by clinicians in the future, in order to predict, in combination with other clinical and biological markers, the risk for preeclampsia in pregnant women. Methods As the major source for MBG is located in the parotid glands of the Bufo Marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. A pre-extraction procedure was elaborated to concentrate and clean the plasma sample prior to its analysis. A LC–MS based assay designed to determine MBG in human plasma is being optimized, giving us the opportunity to investigate MBG in non-pregnant healthy volunteers plasma as well as in early pregnant women plasma. Results and discussion Pure MBG has been successfully extracted from the B. Marinus toad venom and the identity of the compound has been confirmed. An extraction procedure for MBG from plasma has been set up by use of solid phase extraction cartridges. Preliminary results allowed us to authenticate the presence of MBG by LC–MS/MS in non-pregnant women as well as in early pregnancy. Further optimization and validation of the LC–MS assay are needed to quantify MBG plasma levels. However, these pioneering observations, are giving the clinicians a promising perspective for early preeclampsia risk assessment in pregnant women.

Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis.

Introduction: Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion...

Objectives Unbiased metabolite biomarker discovery has revealed that combinations of blood-borne metabolites have the potential to predict preeclampsia accurately at ca. 15 weeks of gestation (Kenny et al., 2010). Our aim is to deploy a dedicated translational effort bringing the merits of de-novo biomarker research to health care providers and patients. Methods To exploit the widespread availability of quadrupole mass spectrometers (QqQ-MS) in clinical laboratories world-wide, a platform migration to QqQ-MS was performed. To support the further refinement of the metabolites-based pre-eclampsia prediction algorithm, and the technical and clinical validation of the test, access to appropriate patient samples from prospective cohorts is warranted. A public-private partnership involving supranational government funding, clinicians and dedicated small and medium sized companies collaborated to establish a pregnancy biobank. Results Thus far, a simple metabolite extraction and a targeted LC-QqQ-MS approach using stable isotope labelled metabolites for relative quantification has been developed. The (semi-)quantitative analysis of circa 40 metabolites with very disparate physicochemical characteristics is achievable in a single 10 min run. The company is engaged in 2 dedicated public-private initiatives, i.e. SCreening fOr Pregnancy Endpoints (SCOPE) (North et al., 2011) and IMproved Pregnancy Outcomes by Early Detection (IMPROvED) (Navaratnam et al., 2013). These collaborations are instrumental for the further clinical and technical validation of the test under development. Conclusions Development of a dedicated translational effort to further exploit newly discovered biomarkers relevant to the prediction of preeclampsia has already resulted in an analytical method ready for testing in clinically relevant patient cohorts. Public–private biobanking efforts have proved to be a cost-efficient means enabling both additional basic biomarker research and progression of biomarkers to market. Disclosures: L. Bond: employee Metabolomic Diagnostics. C. Nolan: employee Metabolomic Diagnostics. K. Hyland: employee Metabolomic Diagnostics. C. Lenaerts: None. P.N. Baker: Commercial Interest: Metabolomic Diagnostics. L.C. Kenny: Commercial Interest: Metabolomic Diagnostics. R. Tuytten: employee Metabolomic Diagnostics.