Utility of Europium ion characteristic peak for quantitation of Fenoterol hydrobromide and Salmeterol xinafoate in different matrices; application to stability studies.

A simple selective luminescent dependent approach was established for quantitation of two selective ß2 agonists namely; Fenoterol hydrobromide (FEN) and Salmeterol xinafoate (SAL). This approach utilizes the capability of the cited drugs to undergo a complexation reaction with Europium ion (Eu3+) in the presence of 1,10-phenanthroline as a co-ligand. The resultant complex leads to a hypersensitive transition and enhancement of the Eu3+ emission peak at 615nm (279nm excitation). Under the optimized conditions, the rectilinear concentration plots of both drugs were (70-1500ngmL-1) and (100-2000ngmL-1) with limit of quantitation 51.3 and 84.4ngmL-1 for FEN and SAL, respectively. The luminescence properties of the complex and its optimum formation conditions were carefully investigated according to the regulations of ICH and the method was successfully applied in plasma. The good accuracy and selectivity of the suggested method allowed extending the proposed protocol into stability study of the cited drugs.

New enhanced spectrofluorimetric approach for picogram detection of Fenoterol hydrobromide through Von Pechman synthesis of coumarins.

A new, specific, precise and very sensitive spectrofluorimetric methodology has been established and approved for determination of Fenoterol hydrobromide (FEN) in its pharmaceutical forms and spiked plasma. The strategy utilized the phenolic nature of FEN and its capacity to undergo Von Pechman synthesis of coumarin. In this study, Fenoterol hydrobromide reacts with ethyl acetoacetate in presence of concentrated sulfuric acid to form an extremely fluorescent coumarin derivative measured at 480 nm (λex: 420 nm). Different reaction variables affecting development and stability of the formed coumarin derivative were precisely examined and enhanced to guarantee greatest sensitivity of the strategy. The recommended procedure was found to obey Beer's law in concentration range of (300-2000) pg mL-1 with quantitation limit 130 pg mL-1, revealing high sensitivity of the suggested method. The proposed procedure was completely examined and approved through the ICH guidelines and was efficiently applied for the determination of the cited drug in spiked plasma and its dosage forms.

Development and validation of a sensitive LC-MS/MS method for the determination of fenoterol in human plasma and urine samples.

Due to the lack of sensitivity in current methods for the determination of fenoterol (Fen), a rapid LC-MS/MS method was developed for the determination of (R,R')-Fen and (R,R';S,S')-Fen in plasma and urine. The method was fully validated and was linear from 50pg/ml to 2000pg/ml for plasma and from 2.500ng/ml to 160ng/ml for urine with a lower limit of quantitation of 52.8pg/ml in plasma. The coefficient of variation was <15% for the high QC standards and <10% for the low QC standards in plasma and was <15% for the high and low QC standards in urine. The relative concentrations of (R,R')-Fen and (S,S')-Fen were determined using a chirobiotic T chiral stationary phase. The method was used to determine the concentration of (R,R')-Fen in plasma and urine samples obtained in an oral cross-over study of (R,R')-Fen and (R,R';S,S')-Fen formulations. The results demonstrated a potential pre-systemic enantioselective interaction in which the (S,S')-Fen reduces the sulfation of the active (R,R')-Fen. The data suggest that a non-racemic mixture of the Fen enantiomers may provide better bioavailability of the active (R,R')-Fen for use in the treatment of cardiovascular disease.

Semi-automated in vivo solid-phase microextraction sampling and the diffusion-based interface calibration model to determine the pharmacokinetics of methoxyfenoterol and fenoterol in rats.

In vivo solid-phase microextraction (SPME) can be used to sample the circulating blood of animals without the need to withdraw a representative blood sample. In this study, in vivo SPME in combination with liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to determine the pharmacokinetics of two drug analytes, R,R-fenoterol and R,R-methoxyfenoterol, administered as 5 mg kg(-1) i.v. bolus doses to groups of 5 rats. This research illustrates, for the first time, the feasibility of the diffusion-based calibration interface model for in vivo SPME studies. To provide a constant sampling rate as required for the diffusion-based interface model, partial automation of the SPME sampling of the analytes from the circulating blood was accomplished using an automated blood sampling system. The use of the blood sampling system allowed automation of all SPME sampling steps in vivo, except for the insertion and removal of the SPME probe from the sampling interface. The results from in vivo SPME were compared to the conventional method based on blood withdrawal and sample clean up by plasma protein precipitation. Both whole blood and plasma concentrations were determined by the conventional method. The concentrations of methoxyfenoterol and fenoterol obtained by SPME generally concur with the whole blood concentrations determined by the conventional method indicating the utility of the proposed method. The proposed diffusion-based interface model has several advantages over other kinetic calibration models for in vivo SPME sampling including (i) it does not require the addition of a standard into the sample matrix during in vivo studies, (ii) it is simple and rapid and eliminates the need to pre-load appropriate standard onto the SPME extraction phase and (iii) the calibration constant for SPME can be calculated based on the diffusion coefficient, extraction time, fiber length and radius, and size of the boundary layer. In the current study, the experimental calibration constants of 338.9±30 mm(-3) and 298.5±25 mm(-3) are in excellent agreement with the theoretical calibration constants of 307.9 mm(-3) and 316.0 mm(-3) for fenoterol and methoxyfenoterol respectively.

Comparison and validation of calibration methods for in vivo SPME determinations using an artificial vein system.

The success of in vivo solid phase microextraction (SPME) depends significantly on the selection of calibration method. Three kinetic in vivo SPME calibration methods are evaluated in this paper: (1) on-fibre standardization (OFS), (2) dominant pre-equilibrium desorption (DPED), and (3) the diffusion-based interface (DBI) model. These are compared in terms of precision, accuracy, and ease of experimental use by employing a flow device simulating an animal circulatory system. In addition, the kinetic calibration methods were validated against established SPME equilibrium extraction (EE) external calibration and a conventional sample preparation method involving protein precipitation. The comparison was performed using a hydrophilic drug fenoterol as the analyte of interest. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the determinations. All three kinetic methods compared well with both EE extraction and the conventional method in terms of accuracy (93-119%). In terms of precision, the DBI model had the best precision in whole blood and buffered phosphate saline solution with %RSD similar to the standard techniques (9-15%). DPED had the poorest precision of %RSD (20-30%) possibly due to errors associated with uncertainty in the amount of standard loaded on-fibre and remaining on the fibre after desorption. In addition, incurred errors could result due to the greater number of fibres used in comparison to the other two calibration methods. The precision of the OFS procedure was better than for DPED primarily because the use of multiple fibres is eliminated. In terms of the ease of use for calibration, the DBI model was the simplest and most convenient as it did not require standards once it had been calibrated or the uptake constant was calculated. This research suggests the potential use of DBI model as the best kinetic calibration method for future in-vein blood SPME investigations.

Quantitative determination of fenoterol and fenoterol derivatives in rat plasma using on-line immunoextraction and liquid chromatography/mass spectrometry.

An on-line immunoextraction and liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol in rat plasma. Sample preparation involved immunoextraction of analytes using an antibody raised against R,R'- and R,S'-aminofenoterol that was immobilized onto chromatographic support. LC was performed on a Waters hydrophilic interaction chromatography (HILIC) column (150 mm x 2.1mm), using an isocratic mobile phase of methanol:ammonium acetate (10mM, pH 6.8) (90:10, v/v) at a flow rate of 0.2 ml/min. The MS was operated in the single ion monitoring mode (m/z 304.2 for R,R'-fenoterol, m/z 318.1 for R,R'-methoxyfenoterol, and m/z 339.2 for R,S'-naphthylfenoterol). Optimization of analytes desorption process from the immunoextraction column was performed by factorial analysis and the sample calibration curves were made with spiked rat plasma samples containing 0.5-100 ng/ml of drugs. The cross-selectivity studies of the antibody were determined and the results suggested high selectivities toward R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol. The accuracy of assay was more than 96% while intra- and inter-day precision of assay were less than 12.4%. Stability studies (2h benchtop, freeze/thaw, and autosampler stability) were conducted and the analytes were stable through out studies. The validated method was used to determine the plasma concentration-time profiles of drugs after oral administration to rats of R,R'-fenoterol, R,R'-methoxyfenoterol and R,S'-naphthylfenoterol.

Preparation and in-vivo pharmacokinetic study of a novel extended release compression coated tablets of fenoterol hydrobromide.

The aim of this study was to formulate extended release compression coated core tablets of fenoterol hydrobromide, a selective beta(2) adrenergic receptor agonist, in an attempt to prevent nocturnal asthma. Two hydrophilic polymers viz Kollidon SR, Polyox WSR 303 and a hydrophobic one (Precirol ATO5) were employed. Compression coated tablets were formulated by preparing a core tablet containing 7.5 mg drug and various amounts of polymer and Emcompress then compressed coated with the same polymeric materials. For comparison purpose different matrix tablets were also prepared employing the same polymers. In-vitro release studies were carried out at different pH (1.2 and 6.8). Pharmacokinetics of extended release tablets as well as commercially available immediate release tablets (Berotec) were studied after oral administration to beagle dogs using a new developed LC-MS/MS method with a lower limit of quantification of 1 ng/ml. Fenoterol release from compression coated tablets was significantly lower than matrix tablets. The mechanism of release was changed with the nature and content of polymer. The release pattern of drug from F16 containing 40 mg Kollidon SR divided in the core tablet (15 mg) and the rest in the compressed coat (25 mg) showed a typical zero order release kinetic that could extend drug release >10 h and reasonable time for 75% to be released (t(75)) (8.92 h). When compared to immediate release Berotec tablet the MRT was significantly extended from 7.03 +/- 0.76 to 10.93 +/- 1.25 h (P < 0.001) and HVD(t 50%Cmax) was also significantly extended from 2.71 +/- 0.68 to 6.81 +/- 0.67 h with expected prevention of nocturnal asthma.

HPLC-electrospray mass spectrometric assay for the determination of (R,R)-fenoterol in rat plasma.

A fast and specific liquid chromatography-mass spectrometry method for the determination of (R,R)-fenoterol ((R,R)-Fen) in rat plasma has been developed and validated. (R,R)-Fen was extracted from 125 microl of plasma using solid phase extraction and analyzed on Atlantis HILIC Silica 3 microm column. The mobile phase was composed of acetonitrile:ammonium acetate (pH 4.1; 20mM) (85:15, v/v), at a flow rate of 0.2 ml/min. The lower limit of detection (LLOD) was 2 ng/ml . The procedure was validated and applied to the analysis of plasma samples from rats previously administered (R,R)-Fen in an intravenous bolus.

Determination of fenoterol in human plasma by HPLC with fluorescence detection after derivatization.

A new method for the determination of fenoterol is described, which uses HPLC separation with fluorescence detection. Dobutamine is employed as an internal standard. The separation was achieved on a short reversed phase column with a mobile phase consisting of water, acetonitrile and methanol. Prior to chromatography both analytes are derivatized with 9-chloroformyl-carbazole. Isolation of the analytes from plasma is carried out by liquid-liquid extraction into 2-butanol after protein precipitation with acetonitrile. The method is capable of estimating fenoterol concentrations in the sub-nanogram per ml range with sufficient accuracy and precision. The determination of fenoterol can now be carried out in the average laboratory without radiolabelled material.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

Voltammetric determination of isoxsuprine and fenoterol in dosage forms and biological fluids through nitrosation.

A simple and highly sensitive voltammetric method was developed for the determination of isoxsuprine HCl (I) and fenoterol HBr (II) in dosage forms and biological fluids. The method is based on treatment of the two compounds with nitrous acid followed by measuring the cathodic current produced by the resulting nitroso derivatives. The voltammetric behavior was studied adopting Direct Current (DCt), Differential Pulse (DPP) and Alternating Current (ACt) polarography. Both compounds produced well-defined, diffusion-controlled cathodic waves over the whole pH range in Britton-Robinson buffers (BRb). At pH 11 and pH 9, the values of diffusion-current constants (Id), were 9.4 +/- 0.3 and 7.7 +/- 0.4 for I and II, respectively. The current-concentration plots for I were rectilinear over the range of 0.6-12 microg/ml and 0.1-12 microg/ml in the DCt and DPP modes, respectively. As for II, the range was 1-20 microg/ml and 0.1-20 microg/ml in the DCt and DPP modes, respectively. The minimum detectability (S/N = 2) were 0.02 microg/ml (approximately 6 x 10(-8) M) and 0.01 microg/ml (approximately 2.6 x 10(-8) M) for I and II, respectively, adopting the DPP mode. The proposed method was applied to the determination of both compounds in dosage forms and the results obtained were in good agreement with those obtained using reference methods. The proposed method was further applied to the determination of isoxsuprine in spiked human urine and plasma. The percentage recoveries adopting the DPP mode were 98.84 +/- 1.18 and 99.26 +/- 0.97, respectively.

Postmortem stability and interpretation of beta 2-agonist concentrations.

This paper describes a series of stability and redistribution studies aimed at understanding the presence and significance of beta 2-agonists in asthma deaths. Salbutamol and terbutaline were shown to be stable in postmortem blood at 23 degrees C for 1 week, 4 degrees C for 6 months and -20 degrees C for 1 to 2 years. However, fenoterol was shown to degrade at 23 degrees C (83% loss), 4 degrees C (93% loss) and -20 degrees C (66% loss) over the same time. Salbutamol concentrations detected in blood taken at the time of body admission to the mortuary were not significantly different from the concentrations detected in blood taken from the same cases at the time of autopsy (45 h later). This suggests that significant postmortem redistribution of salbutamol is unlikely to occur during this period. Postmortem blood concentrations of at least salbutamol are likely to reflect the concentration of these drugs in the body at the time of death.

Pharmacokinetics of beta2-sympathomimetics at the example of fenoterol and conclusion for the administration.

The pharmacokinetics of fenoterol (salbutamol, terbutaline) after systemic administration (i.v., infusion, nasal administration) can be best described by a three-compartmental model. Nasal administration causes an effect-time profile between that of infusion and inhalation. The effects of nasal administration on the lung function and the heart rate depend on the plasma levels of the beta2-agonist. The absorption rate after inhalation differs individually in a large variability (1-27% of the dosage). After inhalation the effect on the lung function does not depend on the absorption rate nor on the plasma levels of the beta2-agonist. After inhalation the effect on the lung function is three- to fivefold more expressed than predictable from the plasma level. From the pk/pd data it can be assumed, that there are 10-20 fold higher concentrations in the airways than in the plasma. It is assumed that there are structures nearby the beta2-receptor responsible for the long-lasting effect observed after inhalation. These depot structures cannot be reached from the plasma in concentrations needed for bronchodilation under in vivo conditions. In respect to the effect/side effect relations, there is no doubt that inhalation is the optimal solution for administering beta2-agonists. In respect to the effect/side effect relations more frequent administration of small doses seems to be more favourable than high doses inhaled in long periods.

Gas chromatographic-mass spectrometric determination of beta 2-agonists in postmortem blood: application in forensic medicine.

A solid-phase extraction procedure is described for the simultaneous determination of terbutaline, salbutamol and fenoterol in human postmortem whole blood, using gas chromatography-electron impact mass spectrometry. The limit of quantitation in 1 ml of blood was 1 ng/ml for all analytes. A linear response was observed over the concentration ranges tested, covering both low and high concentrations of each drug. The recoveries in postmortem blood were: terbutaline, 88%; salbutamol, 86%; fenoterol, 92%; orciprenaline (internal standard), 86%. Coefficients of variations for both intra-assay precision and inter-assay reproducibility ranged between 2.2 and 13.0% for all analytes. This method is sensitive and selective, and has been applied successfully to over 60 postmortem blood specimens.

Pharmacokinetics of fenoterol in pregnant women.

The beta 2-sympathomimetic drug fenoterol (fenoterol hydrobromide, CAS 1944-12-3, Partusisten) is routinely used to inhibit uterine contractions (tocolysis). Investigations of plasma concentrations of those receiving i.v. or oral tocolysis often show different results, both within particular groups of pregnant women and in comparison with non-pregnant persons. The aim of this study was to determine the pharmacokinetics of fenoterol in pregnant women, an important factor which so far had not been known. Four healthy pregnant women with similar weight and gestational age and all with premature labor were administered a continuous intravenous infusion of 4 micrograms fenoterol/min. During and up to 24 hours after the end of the infusion, venous blood samples were taken in order to determine the fenoterol plasma concentrations by radioimmunoassay. From a steady state concentration (css) of 2242 +/- 391 pg/ml (x +/- S.E.), a non-linear two-phased plasma elimination was seen with half-lives t1/2 of 11.40 min and 4.87 h. The area under the plasma concentration-time curve (AUC0-12h) was 6.27 ng/ml x h. The total clearance (Cltot) was 114.8 l/h. These data are nearly the same as the data already known for healthy non-pregnant (male) volunteers. The deviations which are seen in the plasma concentrations in pregnant women in comparison to non-pregnant persons during or after continuous i.v. infusion can therefore not be caused by differences in the pharmacokinetics. Other factors, however, such as body weight and/or gestational age, might influence the results.

Application of a Nafion-modified carbon paste electrode for the absorptive stripping voltammetric determination of fenoterol in pharmaceutical preparations and biological fluids.

The preconcentration of fenoterol on a Nafion-modified carbon paste electrode and its subsequent determination using differential pulse voltammetry is described. The effect of pH and percentage Nafion concentration on the accumulation behaviour of fenoterol was studied, and accumulation curves, calibration graphs and reproducibility studies at two different Nafion concentrations have been carried out in the range 2.5 x 10(-8)-5.0 x 10(-7) M fenoterol. A limit of detection in aqueous solutions, calculated using a signal-to-noise ratio (S/N) of 3, was 9.0 x 10(-9) M. Application of the electrode to pharmaceutical preparations, without sample pretreatment, resulted in acceptable deviation from the stated concentration (RSD = +/- 3.81%, n = 4). For more complex matrices, a suitable extraction procedure was developed, resulting in recoveries of > 90% (urine) and > 75% (serum).

Fenoterol metabolism in man: sulphation versus glucuronidation.

1. Plasma levels of fenoterol (F) and its conjugate metabolites were determined in healthy female subjects and in pregnant women treated for preterm labour. Sulphate (S) and glucuronide (G) conjugates could be quantified. 2. In the healthy volunteers, AUC of both the metabolites was half that of parent compound (AUC-S/AUC-F: 0.42; 0.14-1.16) (AUC-G/AUC-F: 0.49; 0.18-0.86) during i.v. administration of the drug and was several fold that of parent drug (AUC-S/AUC-F: 116.9; 36.4-353.3, AUC-G/AUC-F: 19.9; 5.1-57.5) after p.o. administration indicating extensive presystemic elimination. 3. In the healthy subjects, the AUC ratio of G:S was 1.1 (0.5-2.6) and 0.16 (0.10-0.27) after i.v. and p.o. administration, respectively, thus indicating that sulphation is the prevailing metabolic pathway in the presystemic elimination. 4. In patients, concentration ratios were used for the analysis. During continuous i.v. treatment, Css-S/Css-F was 3.8 (2.5-4.8) and Css-G/Css-F was 1.5 (0.7-2.1). During p.o. treatment Csstrough S/Csstrough-F was 69.4 (32.1-145.7) and Csstrough-G/Csstrough-F 9.4 (5.6-13.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of fenoterol in pregnant and nonpregnant women.

The pharmacokinetics of the beta 2-adrenergic drug fenoterol, which is used as a tocolytic agent in pregnancy, has been investigated in pregnant (n = 9) and nonpregnant (n = 5) women during a constant rate intravenous infusion. Clearance and mean residence time were found to be 1990 (1879/2220; Median, Q25/Q75) ml/min and 9.2 (8.0/14.0) min in the pregnant and 2126 (1915/2130) ml/min and 16.6 (16.5/32.1) min in the nonpregnant women, respectively. In addition, fenoterol clearance was estimated in 88 women from a single blood sample collected at steady state during IV therapy and the effect of gestational age on clearance was studied. Clearance displayed large interindividual variation. There was no apparent correlation between clearance and gestational age. We conclude that there is no need to adjust the dose on pharmacokinetic grounds in the course of pregnancy.

Plasma assay of salbutamol by means of high-performance liquid chromatography with amperometric determination using a loop column for injection of plasma extracts. Application to the evaluation of subcutaneous administration of salbutamol.

An isocratic high-performance liquid chromatography method with amperometric detection for the assay of plasma salbutamol is described. The plasma extract is injected into the chromatographic system via a loop column. This insures the purification of the injected extracts and allows a simple and rapid liquid-solid extraction procedure. The good reliability, as shown by the low limit of detection (0.5 ng/ml) and a precision ranging between 5 and 10%, has permitted the investigation of a new mode of administration of salbutamol using a portable subcutaneous infusion pump. Our results show that subcutaneous administration yields plasma levels comparable with those obtained after usual intravenous doses.