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

Abstract

Millophyline-V ®, (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) cartridges, 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 N-oxide could lead to an increase the detection period of the parent drug during routine GC/MS screening of post-competition greyhound urine samples.