A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of cyclosporin A (CsA) and creatinine using capillary blood has been developed. Venous and capillary blood samples were taken predose and at C2 from 65 heart and lung transplant recipients (65 × 4 samples). For comparisons, serum creatinine and blood CsA concentrations were measured by the Jaffe and EMIT methods, respectively, using an Olympus AU600 analyzer. For the LC-MS/MS assay, samples were prepared in a 96 × 700-μL well block by adding 10 μL of blood (or serum) to 40 μL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 μL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 μL of the supernatant was injected into the LC-MS/MS system. A Waters 2795 high-performance liquid chromatography (HPLC) system was used to elute a C18 cartridge (3 mm × 4 mm) at 0.6 mL/min with a step gradient of 50-100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. The column was maintained at 55°C, and the retention times were creatinine, 0.4 minutes; ascomycin, 0.98 minutes; and CsA, 1.2 minutes. Cycle time was 2.5 minutes, injection to injection. The analytes were monitored using a Quattro microtandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: creatinine, m/z 114>44; d3-creatinine (IS), m/z 117>47; ascomycin (IS), m/z 809>756; and CsA, m/z 1,220>1,203. Assay characteristics were CsA intraassay CV, 3.6-3.0% (33-1,500 μg/L); CsA interassay CV, 6.7-2.5% (10-5,000 μg/L); LC-MS/MS capillary [CsA] = 0.99 × LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 × EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 μmol/L); interassay CV, 5.7-3.3% (80-590 μmol/L); LC-MS/MS capillary [creatinine] = 0.99 Jaffe plasma [creatinine] -42.6, R = 0.87. Total time for the preparation and analysis of 30 samples was approximately 2 hours. This assay will provide a flexible, robust, and cost-effective solution for the monitoring of CsA and creatinine in transplant recipients with potential applications in pediatric medicine and pharmacokinetic studies, in which frequent sampling is required.
- Tandem mass spectrometry