Is 9β-dehydrohalogenation of betamethasone and dexamethasone hindering the detection of banned co-eluting meprednisone? A reverse-phase chiral liquid chromatography high-resolution mass spectrometry approach

Mass spectral analysis of dexamethasone and betamethasone reveal intense signals at m/z 373.19994 (using a Thermo Q Exactive high-resolution mass spectrometer coupled with Dionex UltiMate 3000 UHPLC + operated in the positive ion mode), matching the signal of meprednisone, the 11-oxo version of methylprednisolone, along with its parent signal; possibly due to dehydrohalogenation of these drugs at MS. The parent mass of meprednisone is exactly same as that of dehydrohalogenated mass of dexamethasone and betamethasone; and are co-eluting, displaying same mass spectra. Specifically when they are administered together, identifying meprednisone (a drug for which there is zero tolerance in some regions of the world), is a great challenge with currently available techniques because it could be easily mistaken for dexamethasone or betamethasone, drugs allowed at certain threshold limits for therapeutic considerations. False negative results could be obtained in conventional reverse-phase chromatography and are liable to be abused; hence, establishing “zero tolerance” limits for these compounds often proves ineffective. In this paper, present an effective and reliable analytical method for simultaneously separating and identifying dexamethasone, betamethasone and meprednisone in equine urine and plasma using chiral liquid chromatography-electrospray ionization-mass spectrometry. From the various columns screened, the Lux i-Cellulose-5 chiral column produced high-quality results with extremely good separation. During this study, it is quite evident that dehydrohalogenation occurs only in the mass ionization source; the compounds are very stable in-vivo/in-vitro and do not break down either on-column or during sample preparation.