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Immunoassay Enhanced LC/MS/MS Method for the Determination of Nucleotides

Immunoassay Enhanced LC/MS/MS Method for the Determination of Nucleotides Van Damme Thomas, Jenny Zhang, Frederic Lynen and Pat Sandra. Presentation Outline. Introduction Oligonucleotides ELISA Experimental work and results Conclusion. Introduction: Oligonucleotides.

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Immunoassay Enhanced LC/MS/MS Method for the Determination of Nucleotides

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  1. Immunoassay Enhanced LC/MS/MS Method for the Determination of Nucleotides Van Damme Thomas, Jenny Zhang, Frederic Lynen and Pat Sandra

  2. Presentation Outline • Introduction • Oligonucleotides • ELISA • Experimental work and results • Conclusion

  3. Introduction: Oligonucleotides Important intracellular second messengers Oligonucleotides studied as potential therapeutic agents for viral infections, diseases,… Linear nucleotides sequences, cyclic nucleotides (cGMP,cAMP), phosphorothioate oligo (PF-ODN),… cyclic nucleotides (cGMP,cAMP),

  4. Conventional quantitative method for nucleotides: ELISA assay Enzyme-linked immunosorbent assay: used extensively to measure nucleotide sequences Allows for the recognition of specific antigens (in this case nucleotides)

  5. Conventional quantitative method for nucleotides: ELISA assay → problems: - high cost of an ELISA kit - not sufficiently specific for al nucleotides of interest ( immunological cross-reactions ) → possible solution: tandem mass spectrometry (LC-MS/MS) or immunoassay + tandem MS

  6. Why Tandem MS (QQQ)? Single Quadrupole (Q) Total Ion Chromatogram → low sensitivity → low selectivity

  7. Parent ↓ Daughter ↓ Why Tandem MS (QQQ)? Triple Quadrupole (QQQ) • High increase in sensitivity, • necessary for heavy samples • Highly selective

  8. Cyclic nucleotides: features Cyclic guanosine 3’,5’-monophosphate (cGMP) • Levels of cGMP tightly regulated: • - synthesis: guanylate cyclase (GC) • - degradation: enzyme phosphodiesterases • Inhibition of PDE isoenzymes • → increase in intracellular cGMP concentration • → potentially linked to therapeutic effects • => cGMP could serve as a mechanistic biomarker • Mononucleotide • m/z 345.2 g/mol • concentration levels in human blood: ~ 0.5ng/ml

  9. Cyclic nucleotides: features Cyclic adenosine 3’,5’-monophosphate (cAMP) • Levels of cAMP regulated: • - synthesis: adenylate cyclase (AC) • - degradation: enzyme phosphodiesterase • Second messenger • → intracellular signal transduction • Mononucleotide • m/z 329.2 g/mol • concentration levels in human blood: ~ 5ng/ml

  10. Development of an LC/MS/MS Method for the Analysis of cGMP & cAMP • Stationary phase: Zorbax SB-C18 column • 3.0mm * 150mm * 3.5µm • Mobile phase: A/ 0,1% Formic Acid • B/ ACN/Water/MeOH 1:4:2 • Negative mode (mass spectrometer) • Chromatographic conditions: • - 0.3 mL/min flow rate • - 0% A → 50% B in 10 min

  11. Instrument used for Analysis: API 2000 ESI – negative mode

  12. Comparison: cGMP ↔ cAMP with C18 column on API2000 system in H2O LOD: 5ng/ml (3times standard deviation) LOD: 3ng/ml → suitable for whole blood analysis → unsuitable for whole blood analysis cGMP cAMP 40pg on column Same conditions MRM-mode

  13. Separation of a cGMP & cAMP mixturein MRM-mode in H2O cAMP → cGMP ↓ 200pg on the column Use an isocratic plateau → baseline separation

  14. Sample Preparation Method: Real Samples Protein precipitation • 1ml of plasma (spiked) • Add 3ml of ACN • Shake (5min) and centrifuge (15min) the sample • 3,8ml aliquot of supernatant dried under nitrogen (10min) • Reconstitution of samples: add 100µl of water to the sample • => preconcentrate sample with factor10 ! • Shake (5min) and analyze by LC/MS/MS 1 2 3 4 5

  15. Analysis of cGMP & cAMP in spiked plasma cGMP cAMP 40pg on column (4ng/ml & 10µl injection)

  16. Analysis of cGMP in blood Expected concentrations in human blood: cGMP: ~ 0.5ng/ml cAMP: ~ 5ng/ml cGMP in blood 50µl injection Insufficient sensitivity

  17. Analysis of cAMP in blood Measured cAMP levels in blood: Patient2 Patient1 Patient3 ~5.6ng/ml ~3.3ng/ml ~3.2ng/ml

  18. Sample Preparation Method:HILIC SPEAlternative to immuno extraction? • Hydrophilic interaction liquid chromatography SPE: • → normal phase conditions • → especially for polar molecules • Try to purify the matrix and preconcentrate the sample • HILIC SPE cartridges were made in-house • → Fill a glass cartridge with 500mg • of Silica 60µm particles

  19. Sample Preparation Method:HILIC SPEAlternative to immuno extraction? Condition Wash Elute Add sample → → → • Condition: 3ml water • Equilibration: 3ml of 10/90 50mM ammoniumformate/ACN • Add sample: 10ml sample (1/9 plasma/ACN) • Elute: 5ml of 50/50 water/ACN • Dry sample (under nitrogen) and reconstitute (100µl)

  20. HILIC SPE: Loading & eluting Breakthrough analysis → no breakthrough when loading 10ml Analysis of the desorped cAMP (40pg) → 73.1% recovery

  21. HILIC SPE: cAMPComparison with protein precipitation Protein precipitation (robustness issues): Recovery: 76.4 % } 40pg cAMP (preconcentration) Protein precipitation + HILIC SPE Recovery: 73.1 % => 10-20 fold preconcentration possible => Alternative for immunoafinity LC

  22. Triple Quadrupole sensitivity: system comparison API2000 ↔ API3000 (more sensitive?) API 3000 API 2000 40pg on column (of standard) 40pg on column (of standard) cAMP Exact same conditions ~ 10 times more sensitive

  23. Triple Quadrupole sensitivity: system comparison API2000 ↔ API3000 (more sensitive?) cGMP API 2000 API 3000 40pg 40pg Exact same conditions ~ 8 times more sensitive

  24. Conclusion • Combination of protein precipitation + HILIC SPE • + API3000 instrument allow for cGMP and cAMP • determination (ppt level) in blood in a routine way

  25. Thank you for your attention

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