Quantitation of Cyclosporin A in Cell Culture Media by Differential Mobility Mass Spectrometry (DMS-MS/MS)
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 wordsCyclosporine Cyclosporine A Differential mobility spectrometry Mass spectrometry DMS DMS-MS Bioavailability Cell permeability
Development of this method was supported by NIH: RO1 CA 069390-16 (Paul Vouros, PI).
- 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.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.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.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
- 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
- 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