Mass spectrometric method for distinguishing isomers of dexamethasone via fragment mass ratio: An HRMS approach

The major challenge in identifying dexamethasone, betamethasone, and paramethasone from a mixture of these corticosteroids is difficulty in achieving an efficient separation. In this study, we aimed to develop an efficient technique to identify these co-eluting isomers based on the mass spectral patterns of them and their corresponding phase II metabolites after electrospray ionization. Fragmentation pathways in tandem mass spectrometry revealed acceptable specificity within the groups of conjugates. The method was validated using individual isomers and mixtures at various compositions. The effects of concentration and collision energies on fragmentation patterns were also studied extensively. Matrix-fortified equine urine and plasma samples were also included so that matrix effects and interferences on fragmentation ratios could be elucidated. Preliminary results using biological samples demonstrated the suitability of this analytical strategy for direct measurement from their fragmentation patterns. Possible fragmentation pathways for each isomer were proposed.