Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals.

Orciprenaline sulphate (ORP) is a direct-acting sympathomimetic with mainly beta-adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0-400.0 ng/ml and 10.0-240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra-sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4-106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.

Screening of beta-2 agonists and confirmation of fenoterol, orciprenaline, reproterol and terbutaline with gas chromatography-mass spectrometry as tetrahydroisoquinoline derivatives.

A new method for a comprehensive screening and confirmation of beta-2 agonists in human urine is presented based on gas chromatography-low-resolution mass spectrometry (GC-MS) using electron impact ionisation (EI). After hydrolysis of the conjugates with beta-glucuronidase/arylsulfatase a derivatisation step with formaldehyde converts fenoterol, orciprenaline, reproterol and terbutaline to one derivative, a tetrahydroisoquinoline, while the other beta-2 agonists remain unchanged. Liquid-liquid extraction and trimethylsilylation follow. The tetrahydroisoquinoline derivatives show good gas chromatographic and mass spectrometric behaviour. The detection limit of these four beta-2 agonists in the screening using low-resolution mass spectrometry is 10 ng/ml of urine. The other beta-2 agonists are detected as parent compounds with the same recovery after sample preparation with and without formaldehyde. The EI mass spectra of the tetrahydroisoquinoline derivatives are presented.

Excretion study of the beta2-agonist reproterol in human urine.

An excretion study of the beta2-agonist 7-[3-[(beta-3,5-trihydroxyphenethyl)amino]-propyl]theophylline (reproterol) in human urine, which is reportedly misused by athletes and horses as a doping agent, is presented. The study was performed after an oral administration of 20 mg of reproterol hydrochloride. The collected urine samples were prepared using the standard anabolic steroid extraction procedure and analyzed by gas chromatography coupled with quadrupole mass spectrometry and, also, with high-resolution mass spectrometry (HRMS). The main reproterol metabolite was found, whereas unchanged reproterol was not detected. The structure of the main metabolite was confirmed by an accurate HRMS measurement of diagnostic ions. Finally, an excretion urine profile of the main metabolite is presented. The mass spectrum of another possible unidentified reproterol metabolite is also reported.

Design and applications of a self-aligning liquid junction-electrospray interface for capillary electrophoresis-mass spectrometry.

A simple self-aligning liquid junction-electrospray interface for coupling a capillary electrophoresis (CE) system to an atmospheric pressure ionization (API) mass spectrometer (CE-MS) was developed. In contrast to previous liquid junction interfaces, the self-aligning liquid junction interface simplifies the precise alignment of the CE capillary and the sprayer needle and uses a positive make-up flow. Several capillary CE-MS applications were run using both the self-aligning liquid junction interface and the widely used sheath flow interface for comparison purposes. The electrospray stability of the self-aligning liquid junction interface is consistently better even when non-volatile electrolyte solutions are used. At first, some band broadening was obtained with the self-aligning liquid junction interface. Experiments with different CE buffer systems suggested that this band broadening was caused by the materials used in constructing the interface. By using a more inert material for the sprayer needle, the self-aligning liquid junction exhibits excellent electrophoretic resolution, comparable sensitivity, and higher signal-to-noise ratios when run under the same conditions as the sheath flow interface.

Relative bioavailability of metaproterenol in humans utilizing a single dose, stable isotope approach.

The relative bioavailability of metaproterenol (3,5-dihydroxy-alpha-[(isopropylamino)methyl]benzyl alcohol) following a single dose (10-mg metaproterenol sulfate tablet) was studied in six normal male volunteers using coadministration of a solution of a deuterated analogue (metaproterenol-d7 sulfate). The bioavailability of the tablet formulation relative to that of the oral solution was 92 +/- 9%, with excellent power at the 5% significance level. Comparison of the coadministration of the labeled and unlabeled metaproterenol sulfate solutions in two subjects after a one-week washout demonstrated the absence of an isotope effect on either absorption or elimination. A GC-MS assay for metaproterenol was developed to measure plasma concentrations resulting from oral administration. The assay was linear over the range of 0.5-8 ng/mL, corresponding to typical plasma metaproterenol concentrations obtained after a single 10-mg oral dose. Accuracy and precision data were obtained at metaproterenol concentrations of 1.0 and 2.0 ng/mL plasma to demonstrate the applicability of the assay for bioavailability studies. Following oral administration, metaproterenol showed peak plasma concentrations of 2.2 to 13 ng/mL at 0.75 to 3.0 h, with a terminal harmonic mean half-life of 2.1 h over the plasma concentration range studied. The renal clearance of 133-158 mL/min for metaproterenol slightly exceeds the glomerular filtration rate in humans.

Isolation and characterization of metaproterenol-3-O-sulfate: a conjugate of metaproterenol in human urine.

Metaproterenol (1-(3,5-dihydroxyphenyl)-2-isopropylaminoethanol) is primarily converted in humans to metaproterenol-3-O-sulfate following oral administration. Ion exchange column chromatography with a gradient of ammonium acetate buffer permitted the isolation of the ammonium salt of metaproterenol-3-O-sulfate from human urine. Treatment of aliquots of the column eluate with purified sulfatase and subsequent HPLC/fluorescence analysis confirmed the presence of metaproterenol. Comparison of the column eluate with a metaproterenol standard by 250-MHz proton-NMR revealed a pattern consistent with monosubstitution of the resorcinol ring. Negative and positive ion fast atom bombardment/mass spectrometry showed the metabolite to have a (M-H)- m/z of 290 and a (M + H)+ m/z ion of 292. These three methods support the structural assignment of metaproterenol-3-O-sulfate. Enzymatic hydrolysis of urine specimens from 29 different subjects with purified beta-glucuronidase as well as beta-glucuronidase-sulfatase mixtures yielded no significant increase in metaproterenol beyond purified sulfatase-treated urine, thus ruling out the presence of a glucuronide of metaproterenol. Approximately 40% of an oral 20-mg dose, given as either a tablet or a solution, was recovered in the urine as metaproterenol-3-O-sulfate. Approximately 5% of the dose was recovered in the unconjugated form. The majority of the dose was excreted over the first 12 hr with a biological half-life of 5-6 hr followed by a slower excretion phase with a half-life of 20 hr.

Biotransformation of reproterol in the intestinal tract of the rat.

The major metabolite of reproterol (Broncho-spasmin) in rat feces is 2-[3-theophyllinyl(7)-propyl]-4,6,8-trihydroxy-1,2,3,4-tetrahydroisoquinoline. It was also found in the bile of orally or intravenously dosed rats in the form of glucuronides. The biotransformation of an oral dose of reproterol to this metabolite occurred mostly in the cecum-colon section of the intestinal tract. Experiments in antibiotic treated rats showed no significant effect of the treatment on the extent of metabolite formation. This metabolite was also formed by incubation of reproterol with cecum-colon homogenates under aerobic as well as anaerobic conditions. The pharmacological action after an oral dose must be attributed to reproterol absorbed as intact form, since the metabolite is inactive.