Quantification of unbound prednisolone, prednisone, cortisol and cortisone in human plasma by ultrafiltration and direct injection into liquid chromatrography tandem mass spectrometry:

BackgroundThe existing methods for quantitative analysis of free corticosteroids require high volume of plasma and laborious extraction processes. Development of ultrafiltration followed by the liquid chromatrography tandem mass spectrometry (LC-MS/MS) method that requires 300 μL of plasma, does not entail any offline extraction and achieves good sensitivity was described.MethodsUnbound corticosteroids were separated by the ultrafiltration of plasma using Microcon® centrifugal filter devices (10,000 Dal nominal molecular weight limit). A 30 μL aliquot of the ultrafiltrate was directly injected into a two-dimensional high-performance liquid chromatography clean-up and separation system coupled with API-4000 mass spectrometer. The clean-up was performed on a Strata-X on-line extraction cartridge. A Zorbax-SB Phenyl, Rapid Resolution HT (2.1 × 100 mm) column was employed to chromatographically resolve cortisol and prednisolone from each other, from cortisone and prednisone as well as from interferences found...