Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry

Low-dose corticosteroids may provide a favorable benefit/risk ratio for many therapeutic applications. However, the extremely low plasma drug concentrations achieved, in conjunction with the insufficient sensitivity/ selectivity of current analytical methods, renders the evaluation of corticosteroid pharmacokinetics (PK) a significant challenge under such conditions. Furthermore, targeted therapeutic strategies involving administration by inhalation or intraocular injection could result in very low but sustained systemic corticosteroid concentrations, which must be quantified to determine potential side effects. Here we describe a robust method for the ultrasensitive quantification of corticosteroids in plasma samples. This was achieved by the combination of a selective solid-phase extraction (SPE) with a highly sensitive capillary LC (microLC)-MS/MS analysis. SPE washing and elution conditions were optimized so that target drugs are selectively extracted from plasma. By eliminating most undesirable compounds from the sample matrix, this selective SPE procedure enabled a high sample loading volume on the microLC column without compromising chromatographic performance and operational robustness and helped to achieve ultralow detection limits for the corticosteroids in plasma. The effect of microLC separation on the signal-to-noise ratio of corticosteroid peaks in plasma samples was investigated. It was found that with sufficient microLC separation, sensitivity was improved because of a decrease in matrix effects and the removal of endogenous interferences. Detection limits of four clinically important corticosteroids (budesonide, dexamethasone, triamcinolone acetonide, and dexamethasone acetate) ranged from 0.2 to 1 pg/mL in plasma, and linearity was good for all drugs in the range of 5-5000 pg/mL. Accuracy was 88-107% and the variation (CV%) was 2.3-11.1%. A limit of quantification (LOQ) of 5 pg/mL was validated for all four compounds. We applied this method to quantify the low levels of triamcinolone acetonide (TACA) in porcine plasma following suprachoroidal administration, which is necessary to estimate systemic drug exposure resulting from this novel clinical approach for treating inflammatory diseases of the eye. TACA in plasma could be quantified at low pg/mL levels for up to 90 days posttreatment. To our knowledge, this is the first practical analytical approach that can monitor plasma corticosteroids after intraocular administration, given the ultralow plasma concentrations achieved. In summary, this strategy enables PK analysis of corticosteroids in treatment regimens that result in extremely low systemic concentrations, and the approach can be extended for the sensitive quantification of other drugs.