Simultaneous quantification of cyclosporin A and its major metabolites by time‐of‐flight secondary‐ion mass spectrometry and matrix‐assisted laser desorption/ionization mass spectrometry utilizing data analysis techniques: Comparison with high‐performance liquid chromatography

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Abstract
Simultaneous quantification of cyclosporin A (CsA) and its major metabolite (AM1) in blood has been achieved using time‐of‐flight secondary‐ion mass spectrometry (TOF‐SIMS) and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS). Previous investigations indicated that spectral interferences exist in the analysis of CsA blood samples by the above methods. In TOF‐SIMS, interference is caused by overlap of the Ag‐cationized internal standard, cyclosporin D (CsD), with the Ag‐cationized metabolite, AM1. To resolve this interference and obtain quantitative information, cross‐correlation analysis was applied to the TOF‐SIMS data. Application of damped non‐linear least squares curve‐fitting was carried out to resolve an interference in the MALDI/TOF‐MS data due to multiple cationization products (i.e. Na and K). Measurement of standard samples indicates that the minimum accuracy (95% confidence level) of the TOF‐SIMS method was better than 9% for CsA and 13% for AM1 using only one standard curve'. Similarly, the minimum accuracy of the MALDI/TOF‐MS method was determined to be 14% for CsA and better than 25% for AM1. Blood samples obtained from transplant patients receiving CsA were analyzed by polyclonal fluorescence polarization immunoassay, high‐performance liquid chromatography (HPLC), and by both TOF‐MS methods. Both TOF‐MS results for CsA and mono‐hydroxylated CsA are in good agreement with the HPLC results.