BACKGROUND: Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. METHODS: CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography--tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 x 3.0 mm; 3- microm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 microL. We used positive-ion electrospray MS to monitor the ammonium adducts of the compounds of interest decomposing under controlled conditions to the most dominant fragments of the individual molecules. Calibration curves used linear least-squares regression with 1/x weighting. RESULTS: Maximum sensitivity was obtained by monitoring fragmentation of the ammonium adducts m/z 1220-->m/z 1203 for CsA and m/z 809-->m/z 765 for ascomycin. Sample throughput, including preparation time, was 30 samples in 1.5 h with an injection-to-injection cycle time of 1.5 min. The calibration curve was linear to 5000 microg/L, with a detection limit of 0.03 microg/L and a limit of quantification of 1 microg/L. Regression analysis [tandem MS method (y) and Emit assay (x)] yielded a slope of 1.09 (+/- 0.03), an intercept of 6.2 (+/- 4.5) microg/L, and S(y/x) = 27 microg/L. CONCLUSIONS: Tandem MS assay is a realistic alternative to immunoassay for the routine monitoring of CsA in transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CsA.
BACKGROUND: Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. METHODS:CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography--tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 x 3.0 mm; 3- microm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 microL. We used positive-ion electrospray MS to monitor the ammonium adducts of the compounds of interest decomposing under controlled conditions to the most dominant fragments of the individual molecules. Calibration curves used linear least-squares regression with 1/x weighting. RESULTS: Maximum sensitivity was obtained by monitoring fragmentation of the ammonium adducts m/z 1220-->m/z 1203 for CsA and m/z 809-->m/z 765 for ascomycin. Sample throughput, including preparation time, was 30 samples in 1.5 h with an injection-to-injection cycle time of 1.5 min. The calibration curve was linear to 5000 microg/L, with a detection limit of 0.03 microg/L and a limit of quantification of 1 microg/L. Regression analysis [tandem MS method (y) and Emit assay (x)] yielded a slope of 1.09 (+/- 0.03), an intercept of 6.2 (+/- 4.5) microg/L, and S(y/x) = 27 microg/L. CONCLUSIONS: Tandem MS assay is a realistic alternative to immunoassay for the routine monitoring of CsA in transplant recipients. Its wide dynamic range has utility for pharmacokinetic studies of CsA.
Authors: Emily A Blumberg; Julia Han Noll; Pablo Tebas; Joseph A Fraietta; Ian Frank; Amy Marshall; Anne Chew; Elizabeth A Veloso; Alison Carulli; Walter Rogal; Avery L Gaymon; Aliza H Schmidt; Tiffany Barnette; Renee Jurek; Rene Martins; Briana M Hudson; Kalyan Chavda; Christina M Bailey; Sarah E Church; Hooman Noorchashm; Wei-Ting Hwang; Carl H June; Elizabeth O Hexner Journal: JCI Insight Date: 2022-06-08