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.

Simultaneous enantioseparation and sensitivity enhancement of basic drugs using large-volume sample stacking.

Simultaneous enantioseparation with sensitive detection of four basic drugs, namely methoxamine, metaproterenol, terbutaline and carvedilol, using a 20-mum ID capillary with native beta-CD as the chiral selector was demonstrated by the large-volume sample stacking method. The procedure included conventional sample loading either hydrodynamically or electrokinetically at longer injection times without polarity switching and EOF manipulation. In comparison to conventional injections, depending on the analyte, about several hundred- and a thousand-fold sensitivity enhancement was achieved with the hydrodynamic and the electrokinetic injections, respectively. The simple method developed was applied to the analysis of racemic analytes in serum samples and better recovery was achieved using hydrodynamic injection than electrokinetic injection.

Rapid and automated determination of the beta2-agonist reproterol in human plasma by atmospheric pressure chemical ionisation high-performance liquid chromatography-tandem mass spectrometry.

An on-line HPLC assay with tandem mass spectrometric detection for the fast and sensitive determination of reproterol in human plasma was developed, utilising a methylated structural analogue as the internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250 microl plasma aliquots, at pH 6.5, allowed a reliable quantification of reproterol down to 400 pg/ml. Injection of 100 microl of plasma extracts onto a 30 mm x 4.6 mm reversed-phase guard column provided retention times ranging from 20 to 30 s for reproterol and the internal standard. The standard curves were linear from 0.2 to 200 ng/ml using weighted linear regression analysis (1/y2). The inter-assay and intra-assay accuracies were -0.9% and +3.2%, exhibiting a precision (C.V.) of +/- 11% and +/- 9.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Quantitative determination of terbutaline and orciprenaline in human plasma by gas chromatography/negative ion chemical ionization/mass spectrometry.

A method for the determination of unconjugated terbutaline and orciprenaline in human plasma is described. The assay is based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry. An inexpensive and rapid method for preparation of stable isotope labelled analogues as well as their use in quantitative gas chromatography/mass spectrometry is shown. A highly efficient sample work-up procedure with product recoveries of more than 95% is presented. The method developed permits quantitative measurement of terbutaline and orciprenaline in human plasma down to 100 pg ml-1, using 1 ml of sample. Plasma levels of terbutaline after oral administration of 5 mg of terbutaline sulphate were estimated.

Plasma levels, heart rate, and blood pressure after intravenous, oral, and aerosol administration of reproterol in man.

Three healthy volunteers received single doses of reproterol (D 1959) by means of intravenous infusion, oral and aerosol application, respectively. Administrations were separated by at least 1 week. Plasma levels were measured by means of high performance liquid chromatography. After infusion, plasma levels showed a steep decline, indicative for a rapid distribution. Even though relatively large amounts of drug were given (up to 540 micrograms), this phenomenon caused reproterol levels to drop to values near or below the limit of reliable quantitation (1 ng ml-1) within 60 to 90 min. After oral administration of one or two tablets (containing 20 mg reproterol HCl per tablet), a very short lag time could be observed, indicating fast absorption. After one tablet, plasma level maxima (of plateau-type) were 5-6 ng ml-1 and 2-3 ng ml-1 in two subjects, respectively. After two tablets, plateau level maxima around 18 ng ml-1 and 9 ng ml-1 were found, respectively. After inhalation of a metered aerosol (two puffs of 500 micrograms each) the drug appeared in plasma within minutes, albeit at very low levels, and usually remained detectable at the sub-nanogram level during the time of the experiment (2 h). Due to the very low levels and to some oscillations in the plasma concentration-time curves, a detailed pharmacokinetic assessment could not be carried out. Effects on heart rate and blood pressure were negligible. Only during the infusion of high doses (540 micrograms) was there an increase in heart rate of about 50 to 120 beats min-1. Other side-effects were also negligible.

Analysis of phenylephrine in plasma: initial data about the concentration-effect relationship.

A simple and sensitive assay for phenylephrine in plasma using HPLC with electrochemical detection is presented. Infusion of phenylephrine hydrochloride 0.5, 1.0, 2.0, and 4.0 micrograms X kg-1 X min-1 for 6 min at each dose level to 9 healthy subjects resulted in mean venous plasma concentrations of phenylephrine of 20, 56, 118 and 308 nM, respectively. The systolic and diastolic blood pressures were increased and the heart rate and venous plasma noradrenaline levels fell with increasing phenylephrine concentrations. There was a more than two-fold interindividual variation in the plasma concentration during infusion of the standardized doses of phenylephrine, with overlap of the concentrations achieved at the different dose levels. Estimates of the sensitivity to phenylephrine showed discrepancies within individuals when expressed as the concentration (PC20) or dose (PD20) required to increase systolic blood pressure by 20 mm Hg. Thus, evaluation of concentration-response curves should be more reliable than of dose-response curves when assessing phenylephrine sensitivity during i.v. infusions.

High-performance liquid chromatographic determination of reproterol in plasma using on-line trace enrichment and amperometric detection with a rotating working electrode.

A method is described to determine nanogram quantities of reproterol in plasma. It consists of deproteinization of the plasma samples, on-line trace enrichment, and liquid chromatographic separation of the compounds brought on the analytical column, coupled with amperometric detection. Reliable quantitation can be done down to levels of 1 ng/ml.