Quantitative determination of fenoterol and fenoterol derivatives in rat plasma using on-line immunoextraction and liquid chromatography/mass spectrometry.

An on-line immunoextraction and liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol in rat plasma. Sample preparation involved immunoextraction of analytes using an antibody raised against R,R'- and R,S'-aminofenoterol that was immobilized onto chromatographic support. LC was performed on a Waters hydrophilic interaction chromatography (HILIC) column (150 mm x 2.1mm), using an isocratic mobile phase of methanol:ammonium acetate (10mM, pH 6.8) (90:10, v/v) at a flow rate of 0.2 ml/min. The MS was operated in the single ion monitoring mode (m/z 304.2 for R,R'-fenoterol, m/z 318.1 for R,R'-methoxyfenoterol, and m/z 339.2 for R,S'-naphthylfenoterol). Optimization of analytes desorption process from the immunoextraction column was performed by factorial analysis and the sample calibration curves were made with spiked rat plasma samples containing 0.5-100 ng/ml of drugs. The cross-selectivity studies of the antibody were determined and the results suggested high selectivities toward R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol. The accuracy of assay was more than 96% while intra- and inter-day precision of assay were less than 12.4%. Stability studies (2h benchtop, freeze/thaw, and autosampler stability) were conducted and the analytes were stable through out studies. The validated method was used to determine the plasma concentration-time profiles of drugs after oral administration to rats of R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol.