Simultaneous and rapid analysis of cyclosporin A and creatinine in finger prick blood samples using liquid chromatography tandem mass spectrometry and its application in C2 monitoring.
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 x 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 x 700-microL well block by adding 10 microL of blood (or serum) to 40 microL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 microL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 microL 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 x 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 degrees 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 microg/L); CsA interassay CV, 6.7-2.5% (10-5,000 microg/L); LC-MS/MS capillary [CsA] = 0.99 x LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 x EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 micromol/L); interassay CV, 5.7-3.3% (80-590 micromol/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.
Keevil
Brian G
BG
Department of Clinical Biochemistry, Wythenshawe Hospital, Manchester, United Kingdom. bkeevil@smuht.nwest.nhs.uk
Tierney
David P
DP
Cooper
Donald P
DP
Morris
Michael R
MR
Machaal
Ali
A
Yonan
Nizar
N
D019540:Area Under Curve; D001800:Blood Specimen Collection; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D003404:Creatinine / Q000097:blood*; D016572:Cyclosporine / Q000097:blood*; D017248:Enzyme Multiplied Immunoassay Technique; D016027:Heart Transplantation / Q000276:immunology; D013058:Mass Spectrometry; D012015:Reference Standards
D016572:Cyclosporine; D003404:Creatinine