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 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.

A nuclear magnetic resonance investigation of the upper airways in ferrets. I. Effects of histamine and methacholine.

Alterations induced in the upper airways of ferrets by intranasal provocation with methacholine (MC) and histamine (HS) were monitored using proton magnetic resonance imaging (MRI) and spin-spin relaxation rate (R2) measurements. Both MC and HS cause a significant increase in the MRI signal intensity and a decrease in R2 in the nasal turbinates. A dose-dependent response is observed for 20 to 315 nmol of HS, with a maximum increase in intensity of ca. 50% occurring above 80 nmol. A single unilateral challenge with MC yields a 62 +/- 3% increase in intensity. Control animals (saline-treated) show little change in image intensity. MC and HS cause decreases in the proton R2 by -27.0 +/- 5.5% and -17.2 +/- 4.3%, respectively. These data are indicative of an accumulation of fluid in the nasal airways. MRI provides an effective means to monitor changes in the nasal airways which occur as a result of pharmacological treatment.

Measurement of histamine in rat bronchoalveolar lavage fluid by high-performance cation-exchange chromatography coupled with post-column derivatization to o-phthaldialdehyde.

To better define involvement of mast cell derived mediators in the pulmonary response to fibrogenic dusts, a rapid and accurate method was required to analyze samples of bronchoalveolar lavage fluid for histamine. Samples of rat lung lavage were analyzed for histamine via high-performance cation-exchange chromatography coupled with post-column derivatization with o-phthaldialdehyde. The fluorescent derivative could be detected to ca. 1 ng/ml of lavage. Recoveries averaged 94.2% with an average relative standard deviation of +/- 5.3%. There were no correlations between amount or fibrogenicity of inhaled dust and subsequent release of histamine into lavage fluid.

Magnetic resonance imaging of congenital hydrocephalus in the rat.

Magnetic resonance imaging (MRI) is an increasingly available technique in clinical medicine for the noninvasive imaging of soft tissues. The purpose of the present study was to demonstrate the potential utility of MRI in experimental toxicology and teratology studies. The progression of severity of prenatally induced hydrocephalus was observed in rat pups from 1 to 4 weeks of age. Pregnant Sprague-Dawley rats were given 0, 15, 30, or 45 mg/kg ethylenethiourea (ETU), po, on Gestation Day 15. The two higher doses have been reported to induce a high incidence of hydrocephalus, which is mild at birth but becomes extensive by 4 weeks of age. The low dose was a no effect level for hydrocephalus. None of the doses of ETU altered birth weight or litter size. Pups from each dose group were imaged serially, on Postnatal Days (PD) 6, 13, 17, and 27, in order to determine the progression in the severity of hydrocephalus. Littermates were also imaged on each of these days, then killed immediately in order to compare the anatomy of the brain with its MR image. Hydrocephalus was detectable in the images from all animals of the 30 and 45 mg/kg dose groups on PD 6, the earliest observation day. At this time, the lateral ventricles were dilated less than 1 mm. Hydrocephalus became increasingly severe, and by 4 weeks of age all of the 45 mg/kg group and approximately half of the 30 mg/kg group had died. The brains of the surviving 30 mg/kg rats were severely hydrocephalic, with little cortex remaining. In all cases, the MR image corresponded precisely with the brain anatomy observed after termination. We have demonstrated that MRI is a useful technique for noninvasively imaging lesions in experimental animals. A number of other potential uses for MRI in toxicology are presented.