Asthma and respiratory symptoms occur with a particularly high prevalence in elite athletes compared with the general population in Western countries. As a result, these athletes have a frequent use of antiasthmatic medications, such as beta2-agonists. According to the World Anti-Doping Agency (WADA), the permitted use of beta2-agonists in elite athletes is limited to the inhalation of salbutamol, salmeterol, and formoterol. Salbutamol and salmeterol in therapeutic, inhaled doses have been permitted since 2010. For salbutamol, a urinary concentration exceeding 1000 ng·mL−1 in a doping test is considered an adverse analytical finding. Earlier, the use of formoterol required a therapeutic use exemption (TUE); however, from 2012, this is no longer the case, and formoterol is now permitted in therapeutic inhalations with a maximum of 36 μg·d−1. Urinary concentrations exceeding 30 ng·mL−1 are regarded as an adverse analytical finding, requiring further investigation (16).
Formoterol is a long-acting beta2-agonist with a rapid onset of action. Maximum bronchodilation is achieved within 2 h, and the effects persist for approximately 12 h (15). The pharmacological effects of formoterol are similar to those of other beta2-agonists. Formoterol is a widely used drug when treating athletes with asthma symptoms. Formoterol is indicated as an add-on therapy to maintenance treatment with inhaled corticosteroids, for the relief of broncho-obstructive symptoms and prevention of exercise-induced symptoms in patients with asthma. The normal dose is 4.5–9 μg once or twice daily. Nine micrograms of formoterol as prophylactic treatment before exercise is recommended in patients with exercise-induced asthma (1).
To our knowledge, no studies have shown any performance-enhancing effects of inhalation of long-acting beta2-agonists (3,14). Today, the general view is that inhaled beta2-agonists in therapeutic doses have no effects on the oxygen uptake in athletes, and therapeutic doses of inhaled salbutamol and salmeterol do not required a TUE.
High doses of systemic beta2-agonists can be used to enhance anaerobic power in humans (4,5,9,10). Accordingly, a cutoff value for a beta2-agonist found in the urine or blood is crucial for distinguishing between regular treatment and doping.
We wanted to illustrate the distribution of formoterol in urine and serum after inhalation of 18 μg as a single dose in asthmatic and healthy individuals and, further, in nonasthmatic individuals after inhalation of 18 μg in repeated doses every second hour for 6 h. The purpose was to evaluate concentrations found in urine after therapeutic use with single dose of formoterol and after repetitive use exceeding the permitted daily dose using the WADA 2012 threshold for formoterol.
METHODS AND MATERIALS
The study was an open-labeled survey. On the study day, all participants were interviewed about respiratory symptoms with a questionnaire. Lung function and fractional exhaled nitric oxide (FeNO) were measured, and a skin prick test was performed. Each participant underwent a methacholine challenge to diagnose or rule out asthma.
The protocol was approved by the local ethics committee (protocol no. H-B-2009-047, Eudract 2009-012039-14) and followed the guidelines of good clinical practice.
For the single-dose administration of formoterol, we included 10 men with asthma (Table 1) and 10 healthy men without asthma (Table 2). For the repeated doses of formoterol, we included another 10 healthy men without asthma (Table 3). Subjects were age 18–45 yr.
All subjects with asthma had asthma symptoms; current doctor-diagnosed asthma and a positive methacholine challenge were among the inclusion criteria for this group.
Healthy subjects had no asthma symptoms and no earlier doctor-diagnosed asthma as well as a negative methacholine challenge.
The Global Initiative for Asthma score was used to determine asthma severity (2). All those with asthma had used a beta2-agonist when necessary for at least 1 yr. Exclusion criteria were as follows: 1) more than 6 h of training per week, 2) morbidities other than asthma and allergy, 3) current smoker or earlier smoker with more than or equal to 10 pack-years, or 4) having had an upper or a lower respiratory tract infection during the 2 wk before the study.
All subjects abstained from using any medicine, including inhaled beta2-agonists, 2 wk before enrollment.
Written informed consent was obtained from all participants.
The medication used in the studies was Oxis Turbohaler (Astra Zeneca, London, Great Britain) 9 μg per dose. One dose contains 9 μg formoterol fumarate dihydrate inhalation powder. On the study day, all participants were instructed in inhalation technique. Participants had two puffs immediately after each other when the trial started, that is, 18 μg formoterol. Those receiving repeated doses had two inhalations at the trial start (T0) and again after 2 h (T2), 4 h (T4), and 6 (T6), giving a total of 72 μg inhaled formoterol more than 6 h.
Blood samples (9 mL) were collected from the medial cubital vein at baseline (T0) and 30 min (T30), 60 min (T1), 120 min (T2), 180 min (T3), 240 min (T4), and 360 min (T6) after the administration of formoterol. Each tube stood for 30 min at room temperature before being centrifuged at 3000 rpm corresponding to 2200 g for 15 min. Serum (3–5 mL) was collected in Nunc tubes and kept frozen until analysis.
The total amount of urine produced by each subject was collected every 4 h during the 12 h. Urine was collected from all subjects before the administration of formoterol (T0), and then after 0–4 h (T4), 4–8 h (T8), and 8–12 h (T12). A 30-mL aliquot was stored frozen until analysis. The 12-h urine sample was collected at home and stored at −18°C until the next day when it was delivered frozen to our department, after which it was stored frozen.
Fractional exhaled nitric oxide.
FeNO was measured according to the recommendations of the American Thoracic Society/European Respiratory Society (1) with an NO analyzer (NIOX MINO®; Aerocrine, Stockholm, Sweden) at flow of 50 mL·s−1.
Lung function measurements.
Spirometry was performed following the American Thoracic Society/European Respiratory Society recommendations (11). Forced expiratory volume in 1 s (FEV1) and forced vital capacity were measured using a 7-L dry wedge spirometer (Vitalograph, Buckingham, UK). Predicted values of FEV1 were based on reference values according to Nysom et al. (12).
On the study day, participants completed questionnaires asking about respiratory and allergic symptoms, use of medicine, hospital referrals, and general practitioner and specialist visits. The questionnaire responses were used to determine the Global Initiative for Asthma scores.
Skin prick test.
Subjects underwent a skin prick test to 10 standard aeroallergens (birch, grass, mugwort, horse, dog, cat, house dust mites [Dermatophagoides pteronyssinus and Dermatophagoides farinae], and molds [Alternaria iridis and Cladosporium herbarium]; Soluprick SQ System; ALKAbelló, Hoersholm, Denmark) (7). Atopy was defined as the development of a wheal at least 3 mm in diameter in response to at least one allergen.
Enrollment in the study required a positive methacholine challenge for subjects with asthma and a negative challenge for healthy subjects. The methacholine challenge was considered positive in cases of a 20% or more decrease in FEV1 from the baseline value with PD20 (cumulative dose of methacholine) 8 μmol.
Urine and blood samples were analyzed at the WADA-accredited Norwegian Doping Control Laboratory in Oslo. All tubes were kept frozen during shipment and until analysis.
All reagents used in the analysis were of analytical grade and all solvents were of HPLC grade. Water was obtained using a Millipore purification system (Millipore, Bedford, MA).
An aliquot of 1 mL urine was diluted with an equal volume of water. An internal standard solution (25 μL, 0.1 ng·μL−1 in methanol) of 13C,d3-formoterol (Alsachim, Strasbourg, France) was added, and the samples underwent enzymatic hydrolysis by Escherichia coli β-glucuronidase (Roche Diagnostics, Mannheim, Germany), followed by solid phase extraction (SPE) on Oasis MCX columns (Waters, Milford, MA). Instrumental analysis was performed by liquid chromatography–tandem mass spectrometryon a Thermo Surveyor liquid chromatography system coupled to a Thermo TSQ Quantum Discovery triple-quadrupole mass spectrometer (Thermo-Fisher Scientific, San Jose, CA). The injection volume was 10 μL. The chromatographic separation was performed on a Thermo Betasil C18 column (150 × 2.1 mm, 3-μm particle size) with mobile phases consisting of water–acetonitrile 95:5 (A) and water–acetonitrile 5:95 (B), both containing 5 mM ammonium formiate. The flow rate was 200 μL·min−1, and the elution used a gradient starting at 100% A for 1 min then decreasing linearly to 30% A for 14 min. It was maintained at 30% A for 2 min before returning to the initial conditions of 100% A to equilibrate for 7 min. The mass spectrometer was operated in positive electrospray ionization mode, with a spray voltage of 4 kV and a capillary temperature of 350°C. Nitrogen was used as sheath and auxiliary gas at flow settings of 40 and 20 arbitrary units, respectively. Argon was used as collision gas at a pressure of 1.0 mtorr.
A six-point calibration curve was prepared in blank urine and covered a linear range of 0.05–7.5 ng·mL−1. A new set of calibrators was prepared for each batch of samples. Urine samples containing formoterol at concentrations exceeding 7.5 ng·mL−1 were appropriately diluted and reanalyzed. In the method validation, the limit of quantification in urine was determined as 0.1 ng·mL−1.
The analytical procedure was the same as described for urine, with minor modifications. The amount of internal standard was reduced (25 μL, 13C,d3-formoterol 0.05 ng·μL−1 in methanol), and the enzymatic hydrolysis step was omitted. To avoid interactions between formoterol and serum proteins, phosphoric acid (50 μL, 85% aqueous solution) was added to the samples. The calibration curve was prepared in Autonorm serum (Sero, Billingstad, Norway), and the calibration range was 0.005–0.25 ng·mL−1. The limit of quantification was determined in the method validation as 11 pg·mL−1. For all samples with a concentration lower than limit of quantification, 0 pg·mL−1 was used as the concentration when analyzing the results.
We used the software program Statistical Package for the Social Sciences (Version 19.0; SPSS Inc., Chicago, IL) for the statistical analysis. Normally distributed continuous variables are expressed as mean (SD). Skewed data are presented as median and the observed range of values (range). Values are corrected for urine specific gravity of 1.020 g·mL−1 before analyses. P < 0.05 was considered statistically significant.
The sample size was based on the article by Pichon et al. (13). Power was set to 90%, the level of significance was 5%, and MIREDIF was twice variance.
The concentration of formoterol in the urine samples is shown in Figures 1 and 2.
After the asthmatic subjects inhaled 18 μg formoterol, the two maximum individual urinary concentrations were measured to 15.2 and 15.9 ng·mL−1 in the samples collected after 0–4 h (T4). The maximum individual urinary concentration in healthy subjects was measured to 11.8 ng·mL−1 in the samples collected after 0–4 h (T4).
For asthmatic participants, the maximum median concentration was found after 0–4 h (T4) and was 7.4 ng·mL−1 with the interquartile range (IQR) of 5.3 ng· mL−1. The maximum median concentration also found after 0–4 h in the healthy subjects was 7.9 ng·mL−1, and the IQR was 3.4 ng·mL−1 (no significant difference between the two groups).
The urinary concentrations of formoterol subsequently decreased in both groups. In asthmatic subjects, the medians after 4–8 h (T8) and 8–12 h (T12) were 3.3 ng·mL−1 and 1.7 ng·mL−1, respectively. The corresponding medians were 2.7 ng·mL−1 and 1.5 ng·mL−1 in the healthy group. There were no significant differences between the two groups.
In the healthy subjects who received repeated inhalations of 18 μg formoterol every second hour, the concentration after 0–4 h (T4) was a median of 12.6 ng·mL−1 with an IQR of 8.3 ng·mL−1. The individual maximum after 0–4 h (T4) was 21.4 ng·mL−1. The individual peak concentration of 25.6 ng·mL−1 was reached after 4–8 h (T8). The maximum median concentration was also found after 4–8 h (T8) with a value of 16.8 ng·mL−1 and IQR of 8.1 ng·mL−1. The concentration in the urine decreased after 8–12 h (T12) to a median of 11.7 ng·mL−1 and an IQR of 7.3 ng·mL−1, 6 h after the last administration of formoterol.
The peak individual concentrations uncorrected for urine specific gravity in healthy individuals receiving repeated inhalations were 19.0 ng·mL−1 after 0–4 h (T4), 20.6 ng·mL−1 after 4–8 h (T8), and 20.7 ng·mL−1 after 8–12 h (T12). After 18 μg formoterol, the maximum uncorrected urine concentration was 18.5 ng·mL−1 (15.2 ng·mL−1 corrected) in asthmatic subjects and 15.9 ng·mL−1 (11.8 ng·mL−1 corrected) in healthy subjects after 4 h.
The median concentrations of formoterol in blood samples are shown Table 4.
For those subjects given a single dose of formoterol, the individual maximum concentration was found at T30, reaching 19 pg·mL−1 for asthmatic subjects and 16 pg·mL−1 for healthy subjects. In the same two groups, all the medians were <11 pg·mL−1 for those with asthma and 11 pg·mL−1 (IQR, 15 pg·mL−1) and afterward <11 pg·mL−1 for those without the median at 30 min (T30).
Only one asthmatic subject receiving a single dose of formoterol showed a measurable concentration after 6 h (13 pg·mL−1). None of the healthy subjects receiving a single dose of formoterol had measurable serum concentration after 6 h.
No significant differences were found between the two groups given a single dose of formoterol.
For healthy subjects with repeated inhalation of 18 μg formoterol, the maximum individual serum concentration was measured at T6 to 35 pg·mL−1. The maximum median concentration was found at T6 with a median of 22.5 pg·mL−1 and an IQR of 7.25 pg·mL−1.
In our study of formoterol concentrations in urine and serum, we found no significant pharmacokinetic differences in the urine of asthmatic and healthy subjects given a single dose of inhaled formoterol. The limit of quantification for formoterol in serum is 11 pg·mL−1. Many serum samples are below the limit of quantification, which makes it difficult to rely on the statistics of serum samples from only a few measurable results.
Overall, our study indicates that when formoterol is taken as a single dose in therapeutic inhalation, its concentration in serum is either undetectable (<11 pg·mL−1) or very low.
Our study adds to the existing literature that there are no significant differences between asthmatic subjects with regular use of beta2-agonists and nonasthmatic individuals with no previous use of beta2-agonists, regarding the pharmacokinetics of formoterol after single administration. It could be hypothesized, however, that the long-term use of beta2-agonists could lead to a change in the metabolism of formoterol. Further studies on the long-term use of formoterol with well-known therapeutic doses of formoterol should be performed to evaluate any changes in the pharmacokinetic distribution of formoterol in urine and serum, which could influence on a doping test.
The WADA Prohibited List (2012) allows a daily use of formoterol of 36 μg (16). If higher doses of inhalation are needed, the athlete must have a TUE. WADA has decided to apply a threshold of 30 ng·mL−1 for formoterol concentrations in urine samples.
According to international recommendations, a maximum of two inhalations of 9 μg formoterol at a time should be used when treating asthma, with a maximum daily dose of 36 μg formoterol. Occasionally, a daily dose of 54 μg formoterol can be used (1).
In this study, 20 subjects were given 18 μg formoterol as a single-dose inhalation. Subsequently, two subjects had urine concentrations of more than 15 ng·mL−1, which is more than half the permitted urine concentration (30 ng·mL−1 formoterol) achieved after only half the permitted daily dose (36 μg formoterol). As a single-dose inhalation, 36 μg formoterol is not a therapeutic asthma treatment according to international recommendations; however, the WADA regulations lack specifications about how the permitted daily dose of 36 μg formoterol should be administered.
The observed concentrations in urine after a single-dose administration of 18 μg formoterol in this study are in agreement with the concentrations observed by Deventer et al. (6), which described a quantitative detection of inhaled formoterol in human urine after inhalation of 18 μg formoterol in 6 healthy subjects.
Ten healthy subjects were given a daily dose of 72 μg formoterol in repeated doses of 18 μg every second hour for 6 h to mimic a full day of competition and use of formoterol “as needed” treatment. This is more than the standard recommended dose and twice the daily dose permitted by WADA without a TUE.
The results from repeated doses suggest that formoterol accumulates in both serum and urine. We have no serum concentrations after 6 h, but the urine samples showed that the urinary concentration peaked after the last administration of formoterol and then decreased in the samples collected from 8 to 12 h. Accordingly, we can assume that formoterol is cleared relatively fast from the body. This makes it difficult to differentiate between regular use and excessive use because it is only during a relative short period after use that the high concentrations can be measured.
Until 2012, testing athletes for formoterol in doping tests was determined by whether formoterol was found in the urine sample. In theory, because of the lack of a threshold value, an athlete with a TUE for formoterol had the possibility of using the medicine in excess to enhance performance. Formoterol is a commonly used asthma medicine in sports, especially in the combination budesonide/formoterol (SMART) (8).
With the 2012 changes of the WADA Prohibited List, any excessive use will be easier to define. The threshold value has been set to 30 ng·mL−1 (16). This study supports this as a rational threshold value. The maximum individual values of formoterol found in urine from the 10 healthy participants after inhalation of twice the permitted daily dose were below the threshold value.
Our data from repeated doses of formoterol show a median of 16.8 ng·mL−1 and an IQR of 8.1 ng·mL−1 after inhalation of 72 μg formoterol. We show that there are substantial interindividual differences and support the existing limit of 30 ng·mL−1 formoterol in a urine sample as a cutoff value to report an adverse analytical found. The athlete should then have the option of being tested in a laboratory to measure the formoterol concentrations when given the recommended dose.
The standard dose in the 2012 WADA Prohibited List is 36 μg formoterol. In this study, healthy participants receiving repeated inhalations of formoterol had accumulated a daily dose of 36 μg formoterol after 2 h (18 μg at T0 and 18 μg at T2). The urine samples T4 therefore reflect the values achieved after the daily recommended doses; 21.4 ng·mL−1 formoterol in urine was the highest individual value at T4. Taking into account the substantial interindividual differences, a threshold of 30 ng·mL−1 formoterol found in a doping test should be high enough to minimize the risk of a false-positive test.
We considered the specific gravity of the urine samples when indicating the concentration of formoterol. The hydration of an athlete can influence concentrations of medicines in the urine, so using the urine’s specific gravity is advantageous in cases of dehydration, which can lead to a higher concentration of formoterol. This was the case for one asthmatic subject in our study, 18.3 ng·mL−1 before correction and 15.2 ng·mL−1 after accounting for the urine specific gravity. The study comprised only men. This should be taken into account when evaluating the results as findings might differ in women.
We found the urine’s concentration of formoterol a straightforward marker of the use of formoterol, and the study supports the 2012 WADA threshold of 30 ng·mL−1 formoterol found in urine samples after relevant therapeutic use of formoterol.
Further adjustment to the WADA guidelines could be the use of urine specific gravity when indicating the concentration of formoterol in urine samples and limitation of different beta2-agonist used by one athlete. To our knowledge, no studies have shown the effects of additive use of different kinds of beta2-agonist used at the same time.
Further studies on formoterol should focus on the long-term use of therapeutic doses (18–54 μg formoterol) to reveal any ensuing accumulation and studies evaluating concentrations in urine after 36 μg formoterol taken as a single-dose inhalation.
The study was supported by research grants from WADA.
The authors declare no conflict of interest.
The results of the present study do not constitute endorsement by American College of Sports Medicine.