Rapid and selective method for norepinephrine in rat urine using reversed-phase ion-pair high-performance liquid...

A rugged, high-throughput HPLC-MS-MS-based method, suitable for quantitation of norepinephrine (NE) in urine, has been developed. A rapid, batch-mode procedure utilizes alumina to isolate NE and its deuterated internal standard from urine. After release of NE, using dilute formic acid, samples are analyzed by isocratic reversed-phase ion-pair HPLC, with electrospray ionization (ESI) and MS-MS detection. The ion-pair reagent, heptafluorobutyric acid, is compatible with the ESI interface and permits use of mobile phases with relatively high methanol content, enhancing ESI sensitivity. Furthermore, no significant drop in sensitivity is observed throughout more than 15 h of instrument operation. The selectivity of this approach permitted simplification of the extraction procedure and reduced run times (under 4 min), making single batch-run sizes of more than 200 samples practical. The lower limit of quantitation is 5 ng per 0.5 ml sample, with analytical recoveries of 97-100% and overall method precision of better than 4% relative standard deviation verified up to 500 ng ml(-1). This method was initially applied to study the diurnal rhythm in sympathetic nervous system activity of spontaneously hypertensive rats.