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

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Background: The 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. Methods: Unbound 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 x 100 mm) column was employed to chromatographically resolve cortisol and prednisolone from each other, from cortisone and prednisone as well as from interferences found in plasma from stable kidney transplant recipients. Results: Intra- and inter-run imprecision and inaccuracy within ± 15% were achieved during a three-batch validation for quality control samples at six concentrations in ultrafiltrate from charcoal-stripped plasma and three concentrations from normal plasma, over a 2000-fold dynamic range. The lower limit of quantification was 0.100 ng/mL for all four corticosteroids. Conclusions: A highly selective, sensitive, simple and robust LC-MS/MS method was developed for the simultaneous quantification of free cortisol, cortisone, prednisolone and prednisone. The performance of the Strata-X on-line extraction cartridge was maintained for over 700 injections. The assay was successfully applied for the analysis of the analytes in over 500 plasma samples from stable kidney transplant recipients.

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Annals of Clinical Biochemistry