A LC-MS assay for identification and quantification of ... · PDF fileHere we report the...
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• Marinobufagenin (MBG), a cardiotonic bufadienolide, is a selective inhibitor of the α 1 subunit of Na + ,K + -ATPase. MBG is mainly known due to its role as the major cardiotonic steroid in the Bufo Marinus venom located in the parotoid gland secretions . • Due to its vasoconstrictive, cardiotonic and natriuretic activities, endogenous MBG is implicated in volume expansion-mediated hypertensive states such as preeclampsia. Increased plasma MBG has been observed in preeclamptic women and a rat model for preeclampsia (PE) [1-3]. The increased MBG production seems to appear prior to the development of the symptoms, leading us to consider 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 MBG plasma levels quantification in human. A LC-MS/MS based assay designed to determine MBG in human plasma is being optimized and focuses on our main target: to reach the lowest limit of quantification. • Currently, only marinobufagenin-like material using poor-specific immunoassays has been found in humans [4,5]. Here we report the identification of MBG in non-pregnant human plasma as well as in a plasma sample obtained from a 15 weeks pregnant woman using a LC-MS/MS assay, opening the 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 Preliminar extraction from Bufo Marinus venom Identification of MBG in toad venom Purification process Chromatographic identification of pure MBG A LC-MS assay for identification and quantification of marinobufagenin in human plasma: a novel approach for preeclampsia risk evaluation C. Lenaerts 1 , L. Bond 2 , R. Tuytten 2 , C. Delporte 3 , P. Van Antwerpen 3 , B. Blankert 1 1 Laboratory 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: [email protected]; 2 Metabolomic Diagnostics, Little Island, Cork, Ireland; 3 Analytical 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 extract from crystallized venom Mass spectrum of the pure MBG stock solution • Given that no MBG standard is commercially available, we needed to develop an effective extraction and purification method to acquire the reference compound. • Crystallized Bufo marinus venom was analyzed in order to confirm the presence of MBG in the toad venom using TLC-MS and LC-MS. After optimization of an exctraction method, the identity of purified MBG has been confirmed by TLC-MS and mass spectrometry. 2) Plasma Extraction process SLE process on 96-well plates has been developped and provides selective MBG and IS extraction from plasma with 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. Using the purified MBG, a sensitive MRM based LC-MS/MS assay was developed for MBG. Preliminar tests showed that MBG could be easily detected at 250 pg/mL using 4 mass transitions. By using 2 specific MRM transitions (a quantifier one and a qualifier one), the LC-MS/MS assay allowed us to detect endogenous MBG in both plasma obtained from healthy 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:H 2 O; 0,1% Formic Acid Flow 0.250 mL/min Internal standard 5α-dihydrotestosterone-d 3 [M+H] + : m/z 294 MRM: 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-d 3, 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 2 nd 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 Metabolomic Diagnostics, allowing the achievement of the LC-MS assay development. Telocinobufagin MBG Bufalin MBG MBG MBG Amounts of enriched plasma tested on several types cartridges Resibufogenin UHPLC-MS/MS chromatogram from enriched extracted human plasma Elution solvents with Phenomenex® Novum SLE 96- well plate 4) LC-MS/MS method development: 2 nd 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 MBG 401→365 401→383 401→339 401→347 401→365 401→347 401→339 The optimization of the assay allowed us to quantify MBG at 50 pg/mL with S/N > 10 and to identify endogenous MBG in a 21 weeks pregnant woman plasma sample after extraction UHPLC - QqQ Mass transitions Fragment ion Specificity 401→ 365 m/z [M-2H 2 O] + ↓ (Vit.D 3 ) 401→ 383 m/z [M-H 2 O] + ↓ (Vit.D 3 ) 401→ 339 m/z [M-?] + ↑↑ 401→ 347 m/z [M-3H 2 O] + ↑ 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
Transcript of 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: [email protected]; 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
