Background: The immunosuppressive drug tacrolimus has complex and unpredictable pharmacokinetics, therefore regular monitoring is required in patients receiving tacrolimus therapy. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring tacrolimus concentrations in whole blood and have compared it with a microparticle enzyme immunoassay.
Methods: For the LC-MS/MS assay, samples were prepared in a 96-deep well microtitre plate by adding 10 micro L of blood to 40 micro L of 0.1 mol/L zinc sulphate solution. Proteins were precipitated by adding 100 micro L acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 20 micro L of the supernatant was injected into the LC-MS/MS system. A C18 cartridge (3 mm x 4 mm) was eluted with a step gradient of 50% to 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid, at 0.6 mL/min. The column was maintained at 55 degrees C.
Results: The retention times were 0.98 min for ascomycin and 0.98 min for tacrolimus. Cycle time was 2.5 min, injection to injection. The analytes were monitored using a Quattro micro trade mark tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: m/z821 > 768 (tacrolimus) and m/z809 > 756 (ascomycin). The limit of quantitation was 0.5 micro g/L and the assay was linear up to 30 micro g/L. Precision of the method, over the concentration range 2.5-15.0 micro g/L, was < 7% within-batch and < 6% between-batch. Total time to analyse 24 samples including result generation was 90 min.
Conclusion: We conclude that the LC-MS/MS method is quick, precise and robust and will provide a fast turn around of results for the transplant physician.