SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

In vitro synthesis and characterisation of three fenoterol sulfoconjugates detected in fenoterol post-administration urine samples.

Fenoterol, a fast-acting ß2-adrenergic agonist, is used in the therapy of obstructive pulmonary diseases and for the inhibition of premature labour obstetrics. Doping control for ß2-agonists, which are prohibited in sports by the World Anti-Doping Agency, is commonly performed by liquid chromatography/mass spectrometry after hydrolysis of phase II metabolites. The continuing development of analytical procedures has led to direct injection of urine samples without sample preparation becoming a viable tool. For the detection of substances without sample preparation, including hydrolysis, detailed information of the phase II metabolism of the substances is essential. In this study, human S9 fractions of different tissues and two recombinant sulfotransferases were investigated for their potential to form fenoterol sulfoconjugates, which were characterised in detail. Two mono-sulfoconjugates and one bis-sulfoconjugate were synthesised and their structures confirmed by liquid chromatography­high-resolution/high-accuracy mass spectrometry. All of the metabolites were identified as esterified phenolic compounds. Excretion studies with orally and inhalatively administered fenoterol proved the occurrence of the sulfoconjugates in vivo. Inhalatively administered fenoterol resulted in the detection of the two monosulfoconjugates in low amounts in urine due to the lower inhalation dose of fenoterol compared to the oral dose. After oral uptake of fenoterol, the two mono-sulfoconjugates and a fenoterol bis-sulfoconjugate were detected in urine. This is the first report of the bis-sulfoconjugate.

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.

Pharmacokinetics and metabolism of (R,R)-methoxyfenoterol in rat.

(R,R)-fenoterol (Fen), a beta(2)-adrenoceptor agonist, is under clinical investigation in the treatment of congestive heart disease. The pharmacokinetics and metabolism of the 4-methoxyphenyl derivative of (R,R)-Fen, (R,R)-MFen, have been determined following intravenous and oral administration to the rat and compared with corresponding results obtained with (R,R)-Fen. Results from the study suggest that (R,R)-MFen can offer pharmacokinetic and metabolic advantages in comparison to an earlier (R,R)-Fen. The oral administration revealed that the net exposure of (R,R)-MFen was about three-fold higher than that of (R,R)-Fen (7.2 versus 2.3 min x nmol ml(-1)), while intravenous administration proved that the clearance was significantly reduced, 48 versus 146 ml min(-1) kg(-1), the T(1/2) was significantly longer, 152.9 versus 108.9 min, and the area under the curve (AUC) was significantly increased, 300 versus 119 min x nmol ml(-1). (R,R)-MFen was primarily cleared by glucuronidation associated with significant presystemic glucuronidation of the compound. After intravenous and oral administration of (R,R)-MFen, (R,R)-Fen and (R,R)-Fen-G were detected in the urine samples indicating that (R,R)-MFen was O-demethylated and subsequently conjugated to (R,R)-Fen-G. The total (R,R)-Fen and (R,R)-Fen-G as a percentage of the dose after intravenous administration was 3.6%, while after oral administration was 0.3%, indicating that only a small fraction of the drug escaped presystemic glucuronidation and was available for O-demethylation. The glucuronidation pattern was confirmed by the results from in vitro studies where incubation of (R,R)-MFen with rat hepatocytes produced (R,R)-MFen-G, (R,R)-Fen and (R,R)-Fen-G, while incubation with rat intestinal microsomes only resulted in the formation of (R,R)-MFen-G.

Rapid analysis of clenbuterol, salbutamol, procaterol, and fenoterol in pharmaceuticals and human urine by capillary electrophoresis.

Capillary electrophoresis (CE) with UV detection for the simultaneous and short-time analysis of clenbuterol, salbutamol, procaterol, fenoterol is described and validated. Optimized conditions were found to be a 10 mmoll(-1) borate buffer (pH 10.0), an separation voltage of 19 kV, and a separation temperature of 32 degrees C. Detection was set at 205 nm. Under the optimized conditions, analyses of the four analytes in pharmaceutical and human urine samples were carried out in approximately 1 min. The interference of the sample matrix was not observed. The LOD (limits of detection) defined at S/N of 3:1 was found between 0.5 and 2.0 mgl(-1) for the analytes. The linearity of the detector response was within the range from 2.0 to 30 mgl(-1) with correlation coefficient >0.996.

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.

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.

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.

Determination of fenoterol and ractopamine in urine by enzyme immunoassay.

Fenoterol and ractopamine are phenethanolamines with beta-adrenergic agonist activity. An enzyme immunoassay (EIA) for these compounds was developed using antibodies raised in a New Zealand white rabbit against fenoterol-bovine serum albumin and fenoterol coupled to horseradish peroxidase (HRP). The calibration graphs of fenoterol and ractopamine showed linearity over the concentration ranges 0.1-5 and 0.2-25 ng ml-1, respectively. Isoxsuprine showed a cross-reactivity of 0.7% while the cross-reactivity of other beta-agonists was < 0.1%. The screening assay was used to detect fenoterol in urine samples obtained from an animal experiment in which male calves were treated with fenoterol (100 micrograms of fenoterol per kg of bodymass per meal for a period of four weeks). Using a direct method, without sample preparation, fenoterol concentrations ranged from 22 to 210 ng ml-1. The mean concentration of fenoterol after extraction in isobutanol was 3.5 times lower compared with the direct method. On applying enzymic hydrolysis in combination with isobutanol extraction, the mean concentration was eight times higher than that obtained when using extraction only. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of fenoterol in most of these samples. However, probably owing to the absence of a proper GC-MS internal standard, the correlation between GC-MS and EIA concentrations was low (r = 0.7976). In general, the concentrations found by the EIA are much higher than those found by GC-MS, which might be caused by the presence of metabolites detected with the EIA. Fenoterol is excreted in urine mostly (about 85%) as glucuronidated-sulfated conjugate. The antibodies partly recognize the conjugated fenoterol, which makes it possible to use a direct screening assay. In blank calf urine the detection limits, mean background +3 times the standard deviation, are 1.3 (fenoterol) and 2.6 ng ml-1 (ractopamine). In bovine urine, however, owing to matrix effects, the detection limits are 20 times higher.

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).

Urinary excretion, osmolarity and electrolytes after bolus-injection of fenoterol in female rabbits.

The effects of bolus injections of 1.0-80.0 micrograms/kg body weight fenoterol on urinary excretion, osmolarity and electrolytes were studied in unanesthetized, water-loaded rabbits. In animals infused initially with isotonic solution over 2 h with 60 ml/h and thereafter over 10 h with 45 ml/h, urine excretion was 538 ml/12 h, sodium excretion was 65.4 mmol/12 h, and potassium excretion was 4.8 mmol/12 h. In animals injected with 5.0-80.0 micrograms/kg body weight fenoterol, a strong antidiuresis occurred, lasting for 2 (10.0 micrograms/kg) to 4 h (80.0 micrograms/kg). Due to the strong antidiuresis, urinary osmolarity was significantly elevated for 2 (10.0 micrograms/kg) to 3 h (80.0 micrograms/kg). The changes of sodium excretion after fenoterol injection were very similar to those of urine excretion. Maximum reduction of sodium excretion was found after injection of 10.0-80.0 micrograms/kg body weight fenoterol, the effect lasting for 1 h (10.0 micrograms/kg) to 4 h (80.0 micrograms/kg). Potassium excretion was significantly reduced after injection of 5.0-80.0 micrograms/kg body weight fenoterol. In contrast to all the other parameters measured, potassium excretion remained significantly reduced until the end of the infusion period in animals treated with 10.0-80.0 micrograms/kg body weight fenoterol and was not dose dependent. Our data presented in this work extend earlier findings in the rabbit in that bolus injection of fenoterol also results in a drastic decrease of urine and electrolyte excretion. The results are discussed with special reference for the management of acute fetal distress with betamimetics and to the development of pulmonary edema that has been shown to occur under therapy with betamimetics on both female rabbits and humans.

Studies on the absorption, distribution, metabolism and excretion of 1-(3,5-dihydroxyphenyl)-1-hydroxy-2-[(4-hydroxyphenyl)isopropylamino]-ethane hydrobromide (Th 1165 a, fenoterol hydrobromide) in mice.

Absorption, distribution, metabolism and excretion of 1-(3,5-dihydroxyphenyl)-1-hydroxy-2-[(4-hydroxyphenyl)-isopropylamino]-ethane labelled with 3H (3H-fenoterol) were investigated in mice. After oral administration, 3H-fenoterol was absorbed quickly, reaching a maximum concentration level within 0.5 h after administration, and decreased thereafter with a half-life of about 2 h. Concerning the distribution in tissues after oral administration, digestive tracts showed the highest radioactivity concentration, with lesser concentrations in the kidneys, trachea and liver. The radioactivity concentrations in the other tissues were lower than or equal to that in blood. The concentration in brain tissue was especially low. 24 h after administration, radioactivity was hardly observed in any of the tissues except for intestinal contents. The rates of excretion in urine and in feces in 72 h were ca. 81% and 16%, respectively, with i.v. administration and ca. 45% and 45%, respectively, with oral administration. Metabolites were investigated in urine, serum, liver and kidneys. With every sample the mother compound was hardly observed at all, and most of what was observed were conjugates of fenoterol. The binding rate of 3H-fenoterol with serum protein was investigated with a gel-filtration method. As a result, the binding rate with serum protein was 3--5%.