RAPID AND SELECTIVE METHOD FOR NOREPINEPHRINE IN RAT URINE USING REVERSED-PHASE ION-PAIR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TANDEM MASS-SPECTROMETRY

A rugged, high-throughput HPLC-MS-MS-based method, suitable for quanti tation of norepinephrine (NE) in urine, has been developed. A rapid, b atch-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 conten t, enhancing ESI sensitivity. Furthermore, no significant drop in sens itivity is observed throughout more than 15 h of instrument operation. The selectivity of this approach permitted simplification of the extr action procedure and reduced run times (under 4 min), making single ba tch-run sizes of more than 200 samples practical. The lower limit of q uantitation is 5 ng per 0.5 mi sample, with analytical recoveries of 9 7-100% and overall method precision of better than 4% relative standar d deviation verified up to 500 ng ml(-1). This method was initially ap plied to study the diurnal rhythm in sympathetic nervous system activi ty of spontaneously hypertensive rats. (C) 1998 Elsevier Science B.V. All rights reserved.