N-Deethylation and N-oxidation of etamiphylline:: identification of etamiphylline-N-oxide in greyhound urine by high performance liquid chromatography-mass spectrometry

Millophyline-V-(R), (etamiphylline camsylate) was administered intramuscularly to two racing greyhounds at a dose of 10 mg kg(-1). Unhydrolysed pre- and post-administration urine samples were extracted using mixed mode solid phase extraction (SPE) cartriges, the basic isolates derivatised as trimethylsilyl ethers and analysed by positive ion electron ionisation gas chromatography-mass-spectrometry, (GC/EI+/MS). The parent drug and one metabolite, N-desethyletamiphylline, were detected in urine for up to 72 h. For semi-quantification, urine samples were extracted on-line using a Prospekt sample handler. The analytes retained on the C-2 SPE cartridge were eluted by the mobile phase directly on to the analytical high performance liquid chromatography column and analysed by positive ion atmospheric pressure chemical ionisation (LC/APCI+) MS in the multiple selective-ion recording mode. A major peak containing both ions (m/z) 280 and (m/z) 252 was observed. Full scan LC/APCI+/MS of the unknown indicated that the ion at (m/z) 280 was formed by the loss of an oxygen atom [MH+ --> (MH+ - O)]. Samples were analysed by positive ion electrospray ionisation LC/MS on two different instruments and the unknown compound was identified as an N-oxide of the tert. nitrogen atom of the 2-(diethylamino)ethyl substituent on N-7 of the theophylline nucleus. This compound has not been reported previously either as an in vivo or in vitro metabolite of etamiphylline in any species. Thermal decomposition of the X-oxide could lead to an increase the detection period of the parent drug during routine GUMS screening of post-competition greyhound urine samples. (C) 2004 Elsevier B.V. All rights reserved.