Simultaneous determination of triamcinolone hexacetonide and triamcinolone acetonide in rabbit plasma using a highly sensitive and selective UPLC-MS/MS method.

An ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was successfully developed and qualified for the simultaneous determination of triamcinolone hexacetonide (TAH) and triamcinolone acetonide (TAA, the active metabolite of TAH) in rabbit plasma. To prevent the hydrolysis of TAH to TAA ex vivo during sample collection and processing, we evaluated the effectiveness of several esterase inhibitors to stabilize TAH in plasma. Phenylmethanesulfonyl fluoride (PMSF) at 2.0 mM was chosen to stabilize TAH in rabbit plasma. The developed method is highly sensitive with a lower limit of quantitation of 10.0 pg/mL for both TAA and TAH using a 300 μL plasma aliquot. The method demonstrated good linearity, accuracy, precision, sensitivity, selectivity, recovery, matrix effects, dilution integrity, carryover, and stability. Linearity was obtained over the range of 10-2500 pg/mL. Both intra- and inter-run coefficients of variation were less than 9.1% and accuracies across the assay range were all within 100 ± 8.4%. The run time is under 5 minutes. The method was successfully implemented to support a rabbit pharmacokinetic study of TAH and TAA following a single intra-articular administration of TAH (Aristospan).