a lc-ms assay for identification and quantification of ... · pdf filehere we report the...

• Marinobufagenin (MBG), a cardiotonic bufadienolide, is a selective inhibitor of the α1 subunit ofNa+,K+-ATPase. MBG is mainly known due to its role as the major cardiotonic steroid in the BufoMarinus venom located in the parotoid gland secretions .

• Due to its vasoconstrictive, cardiotonic and natriuretic activities, endogenous MBG is implicated involume expansion-mediated hypertensive states such as preeclampsia. Increased plasma MBG hasbeen observed in preeclamptic women and a rat model for preeclampsia (PE) [1-3]. The increasedMBG production seems to appear prior to the development of the symptoms, leading us toconsider MBG as one of the potential target in the biomarker panel for PE.

• This hypothesis demonstrates the need for an accurate and sensitive analytical method for MBGplasma levels quantification in human. A LC-MS/MS based assay designed to determine MBG inhuman plasma is being optimized and focuses on our main target: to reach the lowest limit ofquantification.

• Currently, only marinobufagenin-like material using poor-specific immunoassays has been found inhumans [4,5]. Here we report the identification of MBG in non-pregnant human plasma as well asin a plasma sample obtained from a 15 weeks pregnant woman using a LC-MS/MS assay, openingthe perspective of investigating the potential of MBG in preeclampsia risk assessment.

Ref: [1] Vu, H.V., et al., American Journal of Nephrology, 2005. 25(5): p. 520-528. [2] Agunanne, E., et al., Amer J Perinatol, 2011. 28(EFirst): p. 509-514. [3] Lopatin, D.A., et al.,Journal of Hypertension, 1999. 17(8): p. 1179-1187. [4] Abi-Ghanem, D., et al., Journal of Immunoassay and Immunochemistry, 2011. 32(1): p. 31-46. [5] Fedorova, O.V., et al.,Circulation, 2002. 105(9): p. 1122-1127

Preliminarextraction fromBufo Marinus

venom

Identification of MBG in toad

venom

Purification process

Chromatographicidentification of

pure MBG

A LC-MS assay for identification and quantification of marinobufageninin human plasma: a novel approach for preeclampsia risk evaluation

C. Lenaerts1, L. Bond2, R. Tuytten2, C. Delporte3, P. Van Antwerpen3, B. Blankert11Laboratory of Pharmaceutical Analysis , Faculty of Medicine and Pharmacy, UMONS Research Institute for Health Sciences and Technology, University of Mons, place du parc 20, 7000 Mons, Belgium. E-mail: charline.lenaerts@umons.ac.be; 2Metabolomic Diagnostics, Little Island,

Cork, Ireland; 3Analytical platform, Faculty of Pharmacy, ULB, Campus de la Plaine, Brussel,Belgium

Gland venom

Marinobufagenin

MBG

TLC-UV of the organic solution from crystallized

venom

UPLC- MS profile of the organic extractfrom crystallized venom

Mass spectrum of the pure MBG stock solution

• Given that no MBG standard is commercially available, we needed to develop an effectiveextraction and purification method to acquire the reference compound.

• Crystallized Bufo marinus venom was analyzed in order to confirm the presence of MBG in thetoad venom using TLC-MS and LC-MS. After optimization of an exctraction method, the identityof purified MBG has been confirmed by TLC-MS and mass spectrometry.

2) Plasma Extraction process

SLE process on 96-well plates has beendevelopped and provides selectiveMBG and IS extraction from plasmawith a 50% recovery for MBG.

MS/MS Identification of MBG in non-pregnant plasma

MS/MS Identification of MBG in plasma at 15 weeks pregnancy

At present, only MBG-like material has been determined in human samples using poor-specific immunoassays. Usingthe purified MBG, a sensitive MRM based LC-MS/MS assay was developed for MBG. Preliminar tests showed that MBGcould be easily detected at 250 pg/mL using 4 mass transitions. By using 2 specific MRM transitions (a quantifier oneand a qualifier one), the LC-MS/MS assay allowed us to detect endogenous MBG in both plasma obtained fromhealthy non pregnant volunteers and from 3 different 15 weeks pregnant woman (n=3).

LC-MS/MS conditions

Stationary Phase Agilent® Poroshell PFP

Mobile Phase Gradient ACN:H2O; 0,1% FormicAcid

Flow 0.250 mL/min

Internal standard 5α-dihydrotestosterone-d3

[M+H]+: m/z 294MRM: 294 → 258 m/z

Ion source and mode

Agilent® 6490 QQQ;JetStream ESI+

3) MS/MS caracterization in human plasma: 1st elaboration

• We obtained pure MBG as a standard for analytical method development following extraction of MBG from Bufo marinus crystallized venom and subsequent purification• A preliminary SLE clean-up step for MBG and the Internal Standard, 5α-dihydrotestosterone-d3, from human plasma has been set up with an extraction yield of 51% for MBG.• A first sensitive LC-MS/MS assay developed at Metabolomic Diagnostics allowed us to authenticate MBG in human plasma: MBG could be identified in non-pregnant healthy patients as well as in early pregnant (15 weeks) volunteers.As far as we know, these initial findings are the first to highlight MBG by LC-MS/MS using specific MRM transitions as thus far only MBG-like compounds have been reported for human plasma..• The LOQ obtained by the 2nd elaborated method (50 pg/mL) fully satisfies the need for quantification of MBG plasma levels in pregnancy (+/- 100 pg/mL range). The quantification method based on the response factor approach will be validated thanks to the accuracy profiles strategy. A primary observational clinical study in pregnant women with non-pregnant controls is now under design and will allow us to confirm previous results observed in case of PE.

Acknowledgements: We thank the laboratory of Prof. Gerbeaux for its collaboration in the development of the extraction method. We acknowledge the « Fédération Wallonie-Bruxelles » for the funding of the collaboration internship with MetabolomicDiagnostics, allowing the achievement of the LC-MS assay development.

TelocinobufaginMBG

Bufalin

MBG

MBG MBG

Amounts of enriched

plasma tested on several

types cartridges

ResibufogeninUHPLC-MS/MS chromatogramfrom enriched

extractedhuman plasma

Elution solvents with

Phenomenex® Novum SLE 96-

well plate

4) LC-MS/MS method development: 2nd elaboration

1) Extraction of MBG from Bufo marinus venom

MBG standard at 250 pg/mL

MBG

MBG identification in 21 weeks pregnant woman plasma sample

MBG401→365

401→383

401→339

401→347

401→365

401→347

401→339

The optimization of the assayallowed us to quantify MBG at50 pg/mL with S/N > 10 and toidentify endogenous MBG in a21 weeks pregnant womanplasma sample after extraction

UHPLC- QqQ

Mass transitions Fragment ion Specificity

401→ 365 m/z [M-2H2O]+ ↓ (Vit.D3)

401→ 383 m/z [M-H2O]+ ↓ (Vit.D3)

401→ 339 m/z [M-?]+ ↑↑

401→ 347 m/z [M-3H2O]+ ↑

Introduction

Results

We obtained pure MBG• A preliminary SLE clean-

Conclusion

MBG and IS standard solution at 50 pg/mL

401→365

401→347

294→258

401→365

401→347

294→258

401→339

ISTD

MBG

MBG

ISTD

MBG

MBG

MBG

MBG

Salzburg, 2016