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Quantitation of Cyclosporin A in Cell Culture Media by Differential Mobility Mass Spectrometry (DMS-MS/MS)

  • Amol Kafle
  • James Glick
  • Stephen L. CoyEmail author
  • Paul Vouros
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2084)

Abstract

Cell permeability is an important factor in determining the bioavailability of therapeutics that is usually measured by cell culture testing. The concentration of pharmaceutical in a medium such as Hank’s Balanced Salt Solution with HEPES organic buffer (HBSS-HEPES) is measured at a series of time points, making simplicity and high throughput of the analytical method important characteristics. We report an electrospray differential mobility spectrometry mass spectrometry method (nanoESI-DMS-MS) for the rapid determination of cyclosporin A (CsA, cyclosporine) concentration in such a buffer. DMS technology provides gas phase atmospheric pressure ion filtration for small-molecule bioanalytical methods that suppresses interfering ions and reduces chemical noise, without the use of chromatography. This allows simplified sample preparation, fast calibration curve development, and shortened analysis times. It has also been noted that the DMS prefilter can reduce contamination of the mass spectrometer by salts, thereby extending mass spectrometer system uptime.

In the application described here, DMS-MS/MS is applied to cyclosporine A (CsA) in cell medium. Sample preparation is limited to dilution with an ammonium acetate-methanol-water mobile phase and the addition of CsA-d4 internal standard. The isotope ratio data are obtained in DMS-MS MRM mode observing NH3 loss from the ammonium adduct of the two species. A calibration curve with high linearity (R2 = 0.998) is rapidly obtained with nearly zero intercept, while it was found that a liquid chromatography LC-MS method required a preliminary SPE step to obtain a linear calibration curve. The time for data acquisition in the DMS-MS MRM method with flow injection (FIA) or infusion introduction at ESI flow of 400 nL/min is typically 30 s leading to a cycle time of less than 1 min.

Key words

Cyclosporine Cyclosporine A Differential mobility spectrometry Mass spectrometry DMS DMS-MS Bioavailability Cell permeability 

Notes

Acknowledgements

Development of this method was supported by NIH: RO1 CA 069390-16 (Paul Vouros, PI).

References

  1. 1.
    Vera NB, Chen Z, Pannkuk E, Laiakis EC, Fornace AJ Jr, Erion DM, Coy SL, Pfefferkorn JA, Vouros P (2018) Differential mobility spectrometry (DMS) reveals the elevation of urinary acetylcarnitine in non-human primates (NHPs) exposed to radiation. J Mass Spectrom 53:548–559.  https://doi.org/10.1002/jms.4085CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Chen Z, Coy SL, Pannkuk EL, Laiakis EC, Fornace AJ Jr, Vouros P (2018) Differential mobility spectrometry-mass spectrometry (DMS-MS) in radiation biodosimetry: rapid and high-throughput quantitation of multiple radiation biomarkers in nonhuman primate urine. J Am Soc Mass Spectrom 29(8):1650–1664.  https://doi.org/10.1007/s13361-018-1977-zCrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kafle A, Klaene J, Hall AB, Glick J, Coy SL, Vouros P (2013) A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP. Rapid Commun Mass Spectrom 27(13):1473–1480.  https://doi.org/10.1002/rcm.6591CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kafle A, Coy SL, Wong BM, Fornace AJ Jr, Glick JJ, Vouros P (2014) Understanding gas phase modifier interactions in rapid analysis by differential mobility-tandem mass spectrometry. J Am Soc Mass Spectrom 25(7):1098–1113.  https://doi.org/10.1007/s13361-013-0808-5CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Shvartsburg AA (2008) Differential ion mobility spectrometry: nonlinear ion transport and fundamentals of FAIMS. CRC Press, Boca Raton, FL.  https://doi.org/10.1201/9781420051070CrossRefGoogle Scholar
  6. 6.
    Schneider BB, Nazarov EG, Londry F, Vouros P, Covey TR (2016) Differential mobility spectrometry/mass spectrometry history, theory, design optimization, simulations, and applications. Mass Spectrom Rev 35(6):687–737.  https://doi.org/10.1002/mas.21453CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Coy SL, Krylov EV, Schneider BB, Covey TR, Brenner DJ, Tyburski JB, Patterson AD, Krausz KW, Fornace AJ Jr, Nazarov EG (2010) Detection of radiation-exposure biomarkers by differential mobility prefiltered mass spectrometry (DMS-MS). Int J Mass Spectrom 291(3):108–117.  https://doi.org/10.1016/j.ijms.2010.01.013CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Coy SL, Cheema AK, Tyburski JB, Laiakis EC, Collins SP, Fornace AJ (2011) Radiation metabolomics and its potential in biodosimetry. Int J Radiat Biol 87(8):802–823.  https://doi.org/10.3109/09553002.2011.556177CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Campbell JL, Yang AMC, Melo LR, Hopkins WS (2016) Studying gas-phase interconversion of tautomers using differential mobility spectrometry. J Am Soc Mass Spectrom 27(7):1277–1284.  https://doi.org/10.1007/s13361-016-1392-2CrossRefPubMedGoogle Scholar
  10. 10.
    van Breemen RB, Li Y (2005) Caco-2 cell permeability assays to measure drug absorption. Expert Opin Drug Metab Toxicol 1(2):175–185.  https://doi.org/10.1517/17425255.1.2.175CrossRefPubMedGoogle Scholar
  11. 11.
    Yee S (1997) In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man—fact or myth. Pharm Res 14(6):763–766.  https://doi.org/10.1023/a:1012102522787CrossRefPubMedGoogle Scholar
  12. 12.
    Hidalgo IJ, Raub TJ, Borchardt RT (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96(3):736–749CrossRefGoogle Scholar
  13. 13.
    Grès M-C, Julian B, Bourrié M, Meunier V, Roques C, Berger M, Boulenc X, Berger Y, Fabre G (1998) Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line. Pharm Res 15(5):726–733.  https://doi.org/10.1023/a:1011919003030CrossRefPubMedGoogle Scholar
  14. 14.
    Covey TR, Thomson BA, Schneider BB (2009) Atmospheric pressure ion sources. Mass Spectrom Rev 28(6):870–897.  https://doi.org/10.1002/mas.20246CrossRefPubMedGoogle Scholar
  15. 15.
    Keevil BG, Tierney DP, Cooper DP, Morris MR (2002) Rapid liquid chromatography-tandem mass spectrometry method for routine analysis of cyclosporin A over an extended concentration range. Clin Chem 48(1):69–76PubMedGoogle Scholar
  16. 16.
    Krylov EV, Coy SL, Vandermey J, Schneider BB, Covey T, Nazarov E (2010) Selection and generation of waveforms for differential mobility spectrometry. Rev Sci Instrum 81(2):024101.  https://doi.org/10.1063/1.3284507CrossRefGoogle Scholar
  17. 17.
    Mason EA, McDaniel EW (1988) Transport properties of ions in gases. Wiley-Interscience, New York, NY.  https://doi.org/10.1002/3527602852CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Amol Kafle
    • 1
  • James Glick
    • 2
  • Stephen L. Coy
    • 1
    Email author
  • Paul Vouros
    • 1
    • 3
  1. 1.Department of Chemistry and Chemical BiologyNortheastern UniversityBostonUSA
  2. 2.Novartis Institutes for BioMedical ResearchEast HanoverUSA
  3. 3.Barnett Institute of Chemical and Biological AnalysisNortheastern UniversityBostonUSA

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