Separation and Identification of the Enantiomeric Doping agent Racemorphan in Equine Urine by Using Normal Phase Chiral-High Resolution Mass Spectrometry

Abstract

In this paper, reported a simple, relatively fast and very sensitive method for the enantiomeric separation of racemorphan (i.e. dextrorphan and levorphanol) by using normal phase chiral liquid chromatography mass spectrometry. The resolution (R = 1.54) of dextrorphan and levorphanol was achieved using the Lux i-amylose-1 column (5 μm, 150 x 4.6 mm) with a mixture of mobile phases consisting of 0.1 % DEA in n-hexane and 0.1 % DEA in 2-propanol (90:10, v/v). To demonstrate the efficacy of the developed method, linearity, accuracy, precision, recovery, sensitivity and effect on matrix in equine urine samples were carried out. For both analytes, the method was linear over the concentration range of 1-50 ng/mL in equine urine. The intra and inter-day precisions expressed as relative standard deviation and the inter-day precisions of dextrorphan, levorphanol were 3.45 % and 3.63 % respectively. The accuracy was in the range of 97-102 %. The limit of detection was 0.1 ng/mL and that of quantification was 0.5 ng/mL for both analytes. Recovery and matrix effect on the analytes were also evaluated. This method is capable of distinguishing racemorphan enantiomers when both of the isomers are simultaneously present in equine urine.