Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 46: 1365-1375, 2000;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergés, R.
Right arrow Articles by de la Torre, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergés, R.
Right arrow Articles by de la Torre, X.
Related Collections
Right arrow Drug Monitoring and Toxicology
(Clinical Chemistry. 2000;46:1365-1375.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Discrimination of Prohibited Oral Use of Salbutamol from Authorized Inhaled Asthma Treatment

Rosa Bergés1,3, Jordi Segura1,3,a, Rosa Ventura1,3, Kenneth D. Fitch2, Alan R. Morton2, Magí Farré1,4, Marta Mas1 and Xavier de la Torre1,4

1 Pharmacology Research Unit, Institut Municipal d’Investigació Mèdica (IMIM), Doctor Aiguader 80, 08003 Barcelona, Spain.

2 Department of Human Movement & Exercise Science, University of Western Australia, Perth, Australia 6907.

3 Universitat Pompeu Fabra, 08005 Barcelona, Spain.

4 Universitat Autònoma de Barcelona, Barcelona, Spain.
a Author for correspondence. Fax 34-93-2213237; e-mail jsegura{at}imim.es


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: The administration of salbutamol is permitted only by inhalation by the International Olympic Committee (IOC) for the management of asthma and exercise-induced asthma in athletes. The establishment of criteria to distinguish between the IOC authorized use (inhaled) and the IOC prohibited use (oral) of salbutamol appeared possible using simultaneous evaluation of variables based on the concentration of nonconjugated enantiomers of salbutamol excreted in urine.

Methods: Urine was collected from asthmatic and nonasthmatic swimmers who had received various preexercise doses of oral (five doses of 4 mg) or inhaled (two doses of 100 µg) salbutamol. Urine was also obtained from subjects who had received the maximum dosage of inhaled salbutamol advisable for competing athletes to provide protection from exercise-induced asthma and treatment of asthma (1600 µg in 24 h, 800 µg being in the last 4 h). All samples were analyzed to determine the total amount of unchanged salbutamol excreted in urine and the ratio between the S and R enantiomers.

Results: The discriminant function D = -3.776 + 1.46 x 10-3 {[S(+)] + [R(-)]} + 1.012 {[S(+)]/[R(-)]} can be used to classify data into two groups, inhaled and oral. The confirmatory criterion suggested (cutoff at D = 1.06, 4 SD from the mean D value of the inhaled distribution) has been verified in different sets of samples showing suspicious concentrations by conventional screening procedures in doping control. An 11.8% false-negative (oral classified as inhaled) rate is assumed with the confirmatory criterion proposed, but virtually no false positives (inhaled classified as oral) are obtained (<1 in 33 000).

Conclusions: The overall procedure recommended is to screen all samples and to apply the confirmation criterion proposed to samples showing free racemic salbutamol concentrations >500 µg/L by gas chromatography-mass spectrometry or free + conjugated racemic salbutamol concentrations >1400 µg/L by ELISA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Salbutamol, a ß2-agonist also known as albuterol, is one of the most widely used asthma medications. It has been on the market for more than 25 years and has been demonstrated safe and effective in the treatment of asthma and as a preventative for exercise-induced asthma (EIA).1 The inhaled route of administration is preferred, whereas oral administration is used mainly with young children who have difficulty in coordinating the inhalation and release of the medication from the metered dose inhaler (MDI). The possible side effects after the administration of salbutamol include muscle tremor, palpitations, muscle cramps during intense exercise, and headache (1).

The List of Prohibited Substances published by the International Olympic Committee (IOC) specifies that the use of salbutamol is only permitted by inhalation in the treatment of asthma or EIA (2). Administration by the oral or parenteral route and the administration of very large inhaled doses are forbidden because of strong adrenergic stimulation and an anabolic-like effect (3)(4) in contrast to administration of common inhaled doses, which have no ergogenic effect (5)(6). Because of the necessity in doping control to distinguish between an authorized and a prohibited use of this ß2-agonist, it is important to develop a urine test with adequate discriminatory capacity. This distinction must extend to the maximum dosage of inhaled salbutamol compatible with treatment of asthma for competing athletes as well as for providing protection from EIA during prolonged exercise.

Salbutamol is excreted in urine as a mixture of the unchanged drug and its conjugated metabolite, mainly sulfate. After oral administration, the majority of the drug is recovered in the urine as the parent compound and the conjugated metabolite, demonstrating that salbutamol is extensively absorbed from the gastrointestinal tract. Approximately 24–33% of an oral dose of racemic salbutamol is excreted unchanged in the urine, and ~48% of the dose is recovered as the conjugated sulfate (7)(8) because of first-pass metabolism. In the lungs, salbutamol is not extensively metabolized, and the proportion of metabolite after inhalation depends mainly on the percentage of the dose that is swallowed after impaction in the mouth and throat and absorbed from the gastrointestinal tract (9).

Salbutamol has a single asymmetric carbon atom, and it is administered as a mixture of two enantiomers: S(+)- and R(-)-salbutamol. As with the majority of other ß-agonists, its therapeutic activity resides predominantly in the R(-) enantiomer with little or no activity attributed to the S(+) enantiomer (10). Recent enantioselective disposition studies of salbutamol after oral administration of the drug have demonstrated that the enantiomers are conjugated at a different rate by the body tissues (11)(12)(13). The active R(-) enantiomer undergoes a higher rate of sulfation, and therefore, after oral intake the nonmetabolized S(+) enantiomer is excreted in a greater proportion than the nonmetabolized R(-). The enantiomeric ratio between S(+) and R(-) after inhalation is close to unity over the first hour after dosing because no metabolism occurs in the lungs. After the first hour, the urinary excretion of the two enantiomers continuously diverges, with larger amounts of the S(+) enantiomer being excreted over the following 12 h (14)(15)(16). This indicates that part of the dose given by inhalation also undergoes enantioselective metabolism. Therefore, it is possible that the proportion of metabolites corresponding to both enantiomers may differ depending on the route of administration.

The establishment of criteria to distinguish between the IOC authorized use (inhaled) and the IOC prohibited use (oral) of salbutamol appeared possible using the simultaneous evaluation of different variables such as the concentration of nonconjugated enantiomers of salbutamol excreted in urine and the ratio between them. The method should be useful to confirm suspicious samples identified after application of conventional screening procedures in doping control (17).

The aim of this study was to compare the discriminatory power of these variables to differentiate between oral and inhaled administration of salbutamol. For this purpose, urine samples obtained in a study involving administration of salbutamol by both routes to nonasthmatic swimmers before routine training sessions were analyzed. Competitive swimmers with asthma were also studied after inhaled salbutamol before a training session. Swimming was selected as the mode of exercise because historically asthmatics have been heavily involved in swimming and the majority of asthmatics who are members of Olympic teams have been in the swimming events (18). Criteria obtained from the experimental data were checked with urine samples obtained in a controlled clinical trial after inhaled and oral administration of different doses of salbutamol, and with samples obtained from doping control.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
study design
This work included three studies designed to examine the differences in the excretion profile between oral and inhaled salbutamol. The protocols for studies 1 and 2 were approved by the Committee for Human Rights at the University of Western Australia (Perth, Australia), and the protocol for study 3 was authorized by the Ethical Committee of Clinical Investigation (CEIC-IMAS, Barcelona, Spain).

Currently, drugs in sport are tested from spot urine samples. Tests are performed either immediately after competition or out of competition, most commonly after a training session. These conditions were simulated as closely as possible in studies 1 and 2.

Study 1 was designed to investigate the possibility to discriminate between 20 000 µg (20 mg) of salbutamol taken orally over a 24-h period (a common oral dosage for anabolic-like effect) and 200 µg of inhaled salbutamol taken immediately before a training session. Study 2 was to determine whether it is possible to distinguish between oral and inhaled salbutamol if the amount of inhaled medication is increased to the maximum advisable dosage for competing asthmatic subjects, i.e., 1600 µg of salbutamol in 24 h, including 800 µg in the last 4 h (19). This is within the manufacturer’s recommendations and allows several doses during a competition lasting 2 or 3 h. Because MDI devices used in conjunction with a "spacer" increase the quantities of drug entering the lungs, it was considered important to determine urinary concentrations of salbutamol after inhalation with and without a spacer. Extrapolation from the urinary concentration to the concentration in blood (central pharmacokinetic compartment) could not be done because the total urine excreted in a given period of time was not collected. In addition, it is known that serial urine concentrations obtained after inhaled salbutamol dosing are not representative of central compartment concentrations (20).

Urine samples from a crossover clinical trial (study 3) involving the administration of single and repeated doses of inhaled and oral salbutamol in random order were also obtained. These samples were used to check the final discriminatory procedure proposed.

specific protocols
Study 1.
Fifteen asthmatic (9 males and 6 females) competitive swimmers and 17 nonasthmatic (10 males and 7 females) recreational swimmers who could comfortably complete 1-h swimming session were recruited. No recreational swimmer who may have been required to undergo a sports doping test was included in this study. All subjects were at least 18 years of age. All subjects signed a written consent form and were required to perform their usual training session after the administration of racemic salbutamol (Ventolin®; Glaxo Wellcome). This procedure simulates a sports drug test.

The asthmatic subjects received only inhaled treatment, and the nonasthmatic group received two treatments, inhaled and oral, in random order and separated by at least 72 h. In the inhaled phase, two inhalations (two puffs) each of 100 µg of salbutamol (200 µg) were administered 5 min before the start of the training session. The oral treatment included five tablets, each containing 4 mg of salbutamol (20 000 µg), that were administered one every 6 h, with the last tablet taken no longer than 2 h before the swimming session. Asthmatic subjects were allowed to continue taking any prescribed medication and any necessary inhaled salbutamol even on the day of testing. The nonasthmatic subjects self-administered the salbutamol using a MDI, whereas the asthmatic swimmers used a spacer with their MDI. The possible side effects from the administration of salbutamol were annotated.

All subjects provided a urine sample before application of any treatment to allow the determination of baseline values. Urine samples were also collected during a 60-min period after the training session and were frozen until analysis.

Study 2.
Sixteen nonasthmatic (10 males and 6 females) volunteers who also participated in study 1 were recruited for the second part of the study.

Sixteen inhalations (8 x 2 puffs) each of 100 µg of salbutamol (1600 µg) were administered over a 24-h period, with 8 of the inhalations (4 x 2 puffs, 800 µg) taken in the last 4 h. Salbutamol was administered through a MDI plus spacer (eight subjects) or via a MDI (eight subjects). The mode of administration was randomly assigned to subjects. Urine samples were collected 60 min after the last inhalation, and a 25-mL aliquot was kept frozen until analysis.

Study 3.
A randomized crossover and non-blind clinical trial was designed to determine the differences in excretion of salbutamol after the administration of single and multiple oral and inhaled doses. The trial included six male healthy volunteers receiving racemic salbutamol under controlled conditions in the absence of exercise. The study consisted of four drug conditions: single and multiple doses of oral salbutamol, and single and multiple doses of inhaled salbutamol. Drug conditions were applied in random order and separated by 1-week washout periods.

For the inhaled phase, subjects received a single dose consisting of four inhalations (4 puffs) each of 100 µg salbutamol (400 µg) or a multiple dose treatment consisting of four doses each of four inhalations (4 x 4 puffs, 1600 µg) administered every 8 h over 24 h. For the oral phase, subjects received a single dose of 4 mg of salbutamol (4000 µg) or a multiple dose consisting of four tablets, each containing 4 mg, administered one every 8 h over 24 h (16 000 µg).

All subjects provided a urine sample before application of treatments to allow the determination of baseline values. Urine samples were collected during a 48-h period after administration of the single dose and during a 72-h period after administration of the multiple doses.

urine analysis
S(+)-Salbutamol and R(-)-salbutamol concentrations in urine were determined using a previously described method (21) involving a solid-phase clean-up procedure followed by a chiral HPLC separation and fluorescence detection. Briefly, urine samples (2 mL) were acidified with 1 mL of 60 g/L acetic acid solution and applied to Bond-Elut CertifyTM (Varian) cartridges preconditioned with 1 mL of methanol and 1 mL of deionized water. The columns were washed with 1 mL of water, 500 µL of 60 g/L acetic acid, and 1 mL of methanol, and dried for 5 min under reduced pressure. Two consecutive elutions (2 mL each) were carried out with a mixture of chloroform and 2-propanol (80:20, by volume) containing 20 mL/L ammonia. The combined eluates were added to 10 µL of a 100 mg/L methanolic atenolol solution (external standard), vortex-mixed, and evaporated to dryness under a stream of nitrogen in a 40 °C water bath. The dried extract was finally reconstituted in 100 µL of dichloromethane-trifluoroacetic acid (218:1, by volume) and vortex-mixed for 1 min; 25 µL was then injected into the HPLC system. A ChirexTM 3022 guard column (30 x 4.0 mm) and a Chirex 3022 analytical column ( 250 x 4.0 mm; Phenomenex) were used. The mobile phase was a mixture of hexane-dichloromethane-methanol-trifluoroacetic acid (250:218:31:1, by volume), the flow rate was 1 mL/min, and the system was operated at room temperature. The effluent was monitored at excitation and emission wavelengths of 230 and 309 nm, respectively. Calibration curves for both enantiomers were prepared daily, and quantification was based on peak-area ratios of the salbutamol enantiomers to the second eluting atenolol enantiomer vs the concentration of compound added.

Final confirmation of the salbutamol enantiomers by mass spectrometry, required in doping control, was accomplished by collection of the fractions containing salbutamol enantiomers after the enantioselective HPLC separation. The mobile phase was evaporated under a stream of nitrogen, and the salbutamol enantiomers were identified by gas chromatographic separation and mass spectrometric detection of the cyclic methylboronate derivatives (22).

stability experiments
Experiments were designed to evaluate the stability of salbutamol and salbutamol sulfate in urine samples after long-term storage. For this purpose, several urine samples collected after oral or inhaled administration of the drug to healthy volunteers were stored for 45 days at different temperatures (-20 °C, 4 °C, room temperature, and 40 °C) and analyzed for the free salbutamol and total (free + conjugated) salbutamol content.

Free S(+)- and R(-)-salbutamol concentrations were evaluated using the enantioselective HPLC procedure described above, whereas total salbutamol was evaluated by the following immunological method (23). The ELISA test Generic Bronchodilators (ELISA Technologies Division, Neogen) was supplied by Labsystems. Sample aliquots of 20 µL were added to each microplate well along with 180 µL of a diluted solution of the conjugate terbutaline-horseradish peroxidase. Wells were incubated by shaking (Heidolph mixer; Labsystems) for 60 min at room temperature, and then were washed three times with 200 µL of diluted washing buffer (Autowash I; Labsystems). After washing, 150 µL of peroxidase substrate solution (K-Blue, proprietary composition) was added to each well, and the plates were incubated for 30 min with shaking to allow color development. The absorbance was determined at 620 nm with an automated microplate reader (Anthos Reader 2001; Bercu Instruments S.L.). A salbutamol calibration curve was analyzed in duplicate with each batch of samples. The following calibration concentrations were used: 0, 0.1, 1, 10, and 100 µg/L racemic salbutamol. Salbutamol calibration curves were calculated using a sigmoidal equation. Urine samples were diluted 1:10, 1:100, or 1:1000 with dilution buffer to obtain a response within the range of the calibration curve. A urine blank and a positive salbutamol control at 10 µg/L were analyzed in each strip of wells.

statistical analysis
The statistical technique that allowed classification of data into groups (inhaled and oral samples) was discriminant analysis. A function involving both the concentration of free salbutamol and the ratio between its enantiomers excreted in urine was obtained using a statistical calculation program (SPSS for Windows, Ver. 7.5.2S). A cutoff value for distinguishing between oral or inhaled administration of the drug was established on the basis of the specificity and the selectivity calculated from the distribution of the discriminant values obtained from the function developed.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Although salbutamol produces fewer cardiac and metabolic effects than other ß2-agonists such as fenoterol, it may cause reductions in serum potassium concentrations and changes in blood pressure and heart rate when administered in high doses (24). In this study, salbutamol concentrations in urine were examined after inhalation of the maximum dosage considered appropriate for asthmatic sportspeople (19). Much higher doses have been recommended by the British Thoracic Society (25) to manage life-threatening asthma. However, athletes that require such large doses of salbutamol suffer such severe asthma that they could not and should not compete. Alternative therapy would be indicated in those cases. Salmeterol, a long-acting ß2-agonist, is permitted in sport and is particularly valuable for events of long duration. The regular daily administration of inhaled corticosteroids (beclomethasone, budesonide, and fluticasone) and/or khellin derivatives (cromoglycate and nedocromil) greatly assist in the control of asthma and EIA in athletes who are tempted to inhale larger quantities of salbutamol. Nevertheless, in 1998, salbutamol was the most commonly identified prohibited and/or restricted drug in sports drug testing (26).

In recent studies involving the detection and quantification of salbutamol in horse urine (27), it was demonstrated that the concentration of free salbutamol was significantly decreased after long-term cold storage. It was assumed that part of the salbutamol exists as an unstable conjugate that may be hydrolyzed during long-term storage or transportation. Therefore, experiments were performed to evaluate the stability of salbutamol and its sulfated conjugate in human urine. Results from four of the samples tested covering a wide range of concentrations are shown in Fig. 1 , where free and total salbutamol concentrations are represented in the different conditions tested. From these studies it was concluded that the concentrations of free salbutamol (individual enantiomers and the addition of both) measured by the enantioselective HPLC procedure and total salbutamol (free + conjugated) measured by the ELISA are rather constant after a 45-day storage period at different temperatures: -20 °C, 4 °C, room temperature, and 40 °C.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. Concentration of free and total salbutamol in urine after a 45-day storage period at different temperatures.

Free salbutamol: {triangleup}, S(+)-salbutamol; {square}, R(-)-salbutamol; •, addition of both. Total salbutamol: {diamondsuit}, free + conjugated. A, -20 °C; B, 4 °C; C, room temperature; D, 40 °C.

Once the stability of salbutamol and its conjugate was verified, all samples from studies 1 and 2 were analyzed using the enantioselective HPLC method with fluorometric detection (21). Concentrations of S(+)- and R(-)-salbutamol excreted in urine samples from the asthmatic and nonasthmatic subjects obtained after oral and inhaled administration of racemic salbutamol were determined.

With the described enantioselective HPLC procedure, salbutamol enantiomers could be separated easily and there was no interference from any of the peaks present in the blank urines obtained for all subjects participating in the studies (n = 68). The retention times of the salbutamol enantiomers were (mean ± SD) 7.78 ± 0.12 and 9.06 ± 0.16 min (n = 10) for S(+)- and R(-)-salbutamol, respectively. Atenolol enantiomers were used as reference for quantification to avoid variability attributable to evaporation of solvents, and their retention times were (mean ± SD) 12.29 ± 0.28 and 13.33 ± 0.33 min (n = 10). Fluorescence detection provided the sensitivity required, with limits of detection of 10.8 and 10.4 µg/L for S(+)- and R(-)-salbutamol, respectively (calculated as 3 SD of the signal-to-noise ratio), and offers great reliability for the assay of large numbers of urine samples (21).

After analyzing the urine samples obtained before the application of any treatment (baseline), we detected no peaks at the retention times of salbutamol enantiomers except for two subjects. These subjects were asthmatics and had baseline salbutamol concentrations because they were allowed to continue taking any necessary inhaled salbutamol even on the day of testing.

Because of the great differences between doses administered orally (mg) and by inhalation (µg), the total concentration of free salbutamol excreted in urine could be useful to differentiate the IOC authorized or prohibited use of salbutamol in sport. The concentrations detected in urine from all subjects are shown in Fig. 2 (top panel). The results indicate that free-salbutamol concentrations >500 µg/L (addition of both enantiomers) are detected in urine after oral administration, whereas after inhalation, concentrations are in general lower than those values. However, some samples obtained after inhaled administration had total free-salbutamol concentrations >500 µg/L and to some extent were similar to those obtained after oral administration. The interindividual variation in urine volume excreted affected the total concentration of free salbutamol; therefore, the discriminatory capacity of this marker alone to fully differentiate between oral and inhaled ingestion is limited.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 2. Concentrations of total free-salbutamol (top) and ratios between free S(+)- and R(-)-salbutamol (bottom) excreted in urine collected from asthmatic and nonasthmatic subjects after oral and inhaled drug administration.

Oral administration: 4 mg every 6 h over 24 h. Inhaled administration: 2 x 100 µg for study 1; 16 x 100 µg over 24 h for study 2. {square}, oral, nonasthmatic; {triangleup}, inhaled, nonasthmatic (study 1); {blacktriangleup}, inhaled asthmatic; {diamond}, inhaled, nonasthmatic (study 2).

The metabolism of salbutamol in humans occurs in the intestine and the liver (28), almost exclusively by sulfate conjugation of the phenolic group (29), and it is highly stereoselective in favor of the R(-) enantiomer (12)(13). The S(+)/R(-) ratios for free salbutamol for the samples analyzed are shown for all subjects in Fig. 2Up (bottom panel). After oral administration, the excretion of free S(+)-salbutamol was favored, and S(+)/R(-) ratios >2.5 were obtained. In these cases, the excretion of the sulfated metabolite dominated, and because of the stereoselective sulfation in favor of the R(-) enantiomer, the excretion of free S(+)-salbutamol was greater than the excretion of free R(-)-salbutamol. On the other hand, after inhaled administration of racemic salbutamol, S(+)/R(-) ratios were in general <2.5. Salbutamol is not extensively metabolized in the lungs, and differences between the excretion of S(+)- and R(-)-salbutamol are lower than after oral administration (30). However, some samples obtained after inhalation gave ratios >2.5, probably because the metabolic behavior after inhalation depends on the proportion of inhaled salbutamol to the proportion swallowed, and in these cases the proportion swallowed is important. In addition, sulfation is competitive between the two enantiomers, and their absolute concentration changes with time influence the value of the ratio observed for the free enantiomers. Therefore, S(+)/R(-) ratios cannot be used alone to fully confirm authorized vs prohibited use of salbutamol.

The simultaneous evaluation of the concentration of unchanged salbutamol and the ratio between the S(+) and R(-) enantiomers measured in urine seem to be useful in establishing authorized or prohibited use of the drug. Plots of total free-salbutamol concentration excreted in urine vs the S(+)/R(-) ratio for samples considered are shown in Fig. 3 . From the results obtained, it can be observed that all urine samples collected after oral administration of racemic salbutamol gave concentrations of free salbutamol >500 µg/L and S(+)/R(-) ratios >2.5. This distribution of values can be clearly separated from that obtained for samples after inhalation. Therefore, our objective was to classify the data into two groups as well as to identify rules for deciding into which class a sample of unknown position should be placed according to a combination of two variables, the total concentration of free salbutamol excreted in urine and the ratio between its enantiomers.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Plot of total free-salbutamol concentration vs the ratio between S(+) and R(-) enantiomers for urine samples collected from asthmatic and nonasthmatic subjects after oral and inhaled drug administration.

Oral administration: 4 mg every 6 h over 24 h. Inhaled administration: 2 x 100 µg for study 1; 16 x 100 µg over 24 h for study 2. {square}, oral, nonasthmatic; {triangleup}, inhaled, nonasthmatic (study 1); {blacktriangleup}, inhaled asthmatic; {diamond}, inhaled, nonasthmatic (study 2); solid line, discriminant function that classifies data into two groups.

The discriminant function developed (31) using the experimental data (n = 45) for samples obtained in studies 1 and 2 after oral ingestion and after inhalation of salbutamol is:

The line that separates the two distributions of points is plotted together with the experimental data set in Fig. 3Up . This analysis showed that the combination of variables defined by the concentration of free salbutamol {[S(+)] + [R(-)]} and the ratio between its enantiomers {[S(+)]/[R(-)]} excreted in urine could distinguish between authorized inhaled and prohibited oral salbutamol better than the individuals markers could. The larger the discriminant score (D), calculated from Eq. 1, the more likely it is that the sample was in the oral group rather than in the inhaled group. Fig. 4 shows the distribution of values of function D calculated for all urine samples obtained after inhalation and oral ingestion of salbutamol in studies 1 and 2. The bottom group of points (Fig. 4 ) shows the distribution for inhaled samples with the vertical lines indicating the mean inhaled D values and distances of 1, 2, 3, and 4 SD from that mean value.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 4. Distribution of D-score values for urine samples collected from asthmatic and nonasthmatic subjects after oral (top) and inhaled (bottom) drug administration.

Oral administration, 4 mg every 6 h over 24 h, study 1. Inhaled administration: 2 x 100 µg for study 1; 16 x 100 µg over 24 h for study 2. Solid vertical line, mean inhaled D value; dashed vertical lines, distances of 1, 2, 3, and 4 SD from the mean value.

To validate a test for use in doping control, a cutoff value should be determined on the basis of the sensitivity (ability to detect a true-positive, or true oral, result) and the specificity (ability to detect a true-negative, or true inhaled, result) as well as the percentage of false positives (inhaled classified as oral: false oral) and false negatives (oral classified as inhaled: false inhaled) obtained. As can be seen in Table 1 , when a cutoff for a D value of -0.33 (2 SD from the mean D value of the inhaled distribution) was chosen, the false oral result rate was nearly 2%, i.e., 2% of the inhaled samples were considered oral. Taking a decision limit for a D value of 1.06 (4 SD from the mean D value of the inhaled distribution), we assumed a false-negative rate of 11.8%, but virtually no false oral results were obtained (<1 in 33 000). This cutoff value implies a better specificity because false-negative results are of less concern in doping control. As can be seen from Fig. 4Up , 15 of the 17 subjects who were administered oral salbutamol would be declared positive for oral administration, with a sensitivity of ~88%.


View this table:
[in this window]
[in a new window]
  		Table 1. Sensitivity, specificity, false-negative, and false-positive (%) results obtained at different cutoff values of the discriminant score D, defined by Eq. 1.

The criterion suggested (4 SD from the mean D value of the inhaled distribution) has been verified in several different sets of real samples. For that purpose, we studied the concentrations of S(+)- and R(-)-salbutamol excreted in urine samples obtained after single and multiple doses of inhaled and oral salbutamol from the six healthy volunteers who participated in the clinical trial (study 3) that showed "suspicious" salbutamol concentrations as defined by conventional screening procedures (17). In Fig. 5 , the distribution of D values for all samples with concentrations higher than the screening cutoff for non-sulfated salbutamol [500 µg/L by gas chromatography–mass spectrometry (GC/MS); Fig. 5 , top panel] and concentrations higher than the screening cutoff for total salbutamol (1400 µg/L free + conjugated compound by ELISA screening; data for two volunteers the bottom panel of Fig. 5 ) are represented. It is interesting to note that the selection of samples by previous screening procedures eliminates those obtained many hours after an inhaled dose that could have a high [S(+)]/[R(-)] ratio while having a very low [S(+)] + R(-)] concentration. If those samples were analyzed only by the confirmatory HPLC procedure, some borderline results for Dmean + 4 SD could be obtained. Moreover, all screening results for suspicious samples collected after competition at the Nagano Winter Olympic Games [Ref. (17) and Ueki M, internal report] with declared inhaled salbutamol were analyzed with the described enantioselective HPLC procedure. As can be seen in Fig. 5 , the confirmatory method allowed classification of all of the inhaled samples into their correct groups (experimental or declared), and no false positives were obtained. Conversely, a doping urine sample from an athlete who had declared in a routine doping test the ingestion of oral salbutamol was also classified according to his declaration. Therefore, a definitive distinction between prohibited oral and authorized inhaled salbutamol in doses that are adequate for all asthmatic athletes to compete can be achieved by HPLC confirmatory urinalysis of suspicious samples selected previously by appropriate screening procedures.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Distribution of D values for urine samples collected after inhalation and oral ingestion of racemic salbutamol in studies 1 and 2 (•), in the controlled clinical trial (study 3; {blacktriangleup}), and during the Nagano Winter Olympic Games ({blacksquare}) that had GC/MS concentrations of non-sulfated salbutamol >500 µg/L (top) or ELISA concentrations of total salbutamol (free + conjugated) >1400 µg/L (bottom) after application of conventional screening procedures in doping control.

A doping sample from an athlete who declared oral salbutamol is included ({diamondsuit}). Solid vertical line, mean inhaled D value; dashed vertical line, distance of 4 SD from the mean value.

The overall procedure recommended in actual sports drug testing is to screen all samples with GC/MS or ELISA procedures and to select for confirmation only those showing free racemic salbutamol concentrations >500 µg/L (GC/MS) or free + conjugated racemic salbutamol concentrations >1400 µg/L (ELISA) (17). The confirmation criteria for a prohibited use of salbutamol would be a discriminant D value (calculated from Eq. 1) after chiral analysis >1.06 (Dmean + 4 SD).

The fact that sulfate metabolism can be saturated by high doses of other drugs, such as acetaminophen (32) or ascorbic acid (33), will need further attention. In such a case, the result of the interaction would be in favor of the athlete because a higher percentage of the drug would go unmetabolized, especially the R(-) enantiomer, and the free [S(+)]/[R(-)] ratio would appear more like dosing via an inhalant. The existence of a group of masking agents in the List of Prohibited Substances may allow inclusion of this possibility if additional research so demonstrates. Self-saturation by salbutamol, if possible, would require much higher doses than those considered physiological and that are not compatible with healthy sports competitors because of the appearance of serious central nervous system side effects (1). These processes, as mentioned above, will not cause false positivity, which is paramount in sports drug testing so that athletes are not falsely accused. The potential effects of some new drugs that affect the absorption of other drugs (e.g., inhibitors of P-glycoprotein) (34) or some disease conditions (viral infections, impaired liver function, lowered liver blood flow) that affect the oral bioavailability of salbutamol by controlling the rate at which the drug is presented to the liver or that affect the metabolic disposition of the enantiomers may be of interest to explain potential aberrant results. It can be also argued that the recent availability of Levalbuterol (the R enantiomer of salbutamol) as a medication in some countries (35) might open the door to its prohibited oral use to escape detection by the present method. The fact that the product is presently available only as a nebulized solution limits to some extent its potential administration by the oral route. In addition, the conclusion that "Levalbuterol appears to have no clinically significant advantage over racemic albuterol" (35) does not foresee its generalized use. Nevertheless, whatever the eventual prevalence of its use, the co-administration of inhaled Levalbuterol together with oral administration of the racemic product might shift the value of the discriminant function toward lower D values with a resulting masking effect, which deserves further studies. In any case, as mentioned above, this situation should never cause an innocent athlete to be falsely accused.

In the proposed methodology, definitive identification of salbutamol enantiomers by MS, usually required in doping control, is accomplished by collection of enantiomers after chiral HPLC separation, derivatization by formation of the cyclic methylboronate, and GC/MS analysis (see Fig. 6 ). The possibility of using the ratios of parent to metabolite enantiomers for detection of the route of administration should be also a topic for discussion. Moreover, the potential use of coupled chiral HPLC-MS (36) should be also investigated.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 6. Analysis of a urine sample containing 971.8 µg/L S(+)-salbutamol and 296.1 µg/L R(-)-salbutamol.

(Top), chiral HPLC chromatogram of the urine sample, collected after oral administration of the drug. (Middle), selected ion monitoring (m/z 230, 272, and 287) of the gas chromatographic peaks for the derivatized eluates containing S(+)- and R(-)-salbutamol enantiomers. (Bottom), mass spectra for the methylboronate derivatives.

As for any decision to be taken by sport governing bodies based on laboratory analytical results in sports drug testing, additional investigations would be prudent before initiating the sanctioning process. In this regard, the need of a certificate by a respiratory or team physician as required for inhaled salbutamol use in the doping regulations, the scrutiny of the data collected on the official forms regarding medications taken by an athlete in the last few days, and his or her clinical asthmatic history (mandatory by some federations or governing bodies) may afford complementary information.


   Acknowledgments
 
Financial support from the IOC is gratefully acknowledged. We thank Dr. S. Pleasance from Glaxo Wellcome (Uxbridge, UK) for the kind donation of pure salbutamol enantiomer calibrators. The collaboration of Dr. M. Ueki from MBC, Inc. (Tokyo, Japan) is gratefully appreciated. We thank S. Berruezo for technical assistance in ELISA analysis and Dr. F. Sanz for assistance in statistical discussions. We also acknowledge Dr. P.N. Roset, E. Menoyo, and C. Hernández for collaboration in the clinical trial.


   Footnotes
 
1 Nonstandard abbreviations: EIA, exercise-induced asthma; MDI, metered dose inhaler; IOC, International Olympic Committee; and GC/MS, gas chromatography–mass spectrometry.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Dolley C, ed. Therapeutic drug, Vol. 2. London: Churchill Livingstone, 1991;51:54pp..
  2. . IOC Medical Commission. List of prohibited substances and methods 1999 IOC Medical Commission Lausanne, Switzerland. .
  3. Martineau L, Horan MA, Rothwell NJ, Little RA. Salbutamol, a ß2-adrenoceptor agonist, increases skeletal muscle strength in young men. Clin Sci 1992;83:615-621.[Medline] [Order article via Infotrieve]
  4. Caruso JF, Signorile JF, Perry AC, LeBlank B, Williams R, Clark M, et al. The effects of albuterol and isokinetic exercise on the quadriceps muscle group. Med Sci Sports Exerc 1995;27:1471-1476.[ISI][Medline] [Order article via Infotrieve]
  5. Morton AR, Papalia SM, Fitch KD. Is salbutamol ergogenic? The effect of salbutamol on physical performance in high-performance non-asthmatic athletes. Clin J Sports Med 1992;2:93-97.
  6. Meeuwisse WH, McKenzie D, Hopkins SR, Road JD. The effect of salbutamol on performance in elite non-asthmatic athletes. Med Sci Sports Exerc 1992;:241161-241166.
  7. Morgan DJ, Paull JD, Richmond BH, Wilson-Evered E, Ziccone SP. Pharmacokinetics of intravenous and oral salbutamol and its sulphate conjugate. Br J Clin Pharmacol 1986;22:587-593.[ISI][Medline] [Order article via Infotrieve]
  8. Hussey EK, Donn KH, Powell JR. Albuterol extended-release products: a comparison of steady-state pharmacokinetics. Pharmacotherapy 1991;11:131-135.[ISI][Medline] [Order article via Infotrieve]
  9. Hindle M, Chrystyn H. Determination of the relative bioavailability of salbutamol to the lung following inhalation. Br J Clin Pharmacol 1992;34:311-315.[ISI][Medline] [Order article via Infotrieve]
  10. Brittain RT, Farmer JB, Marshall RJ. Some observations on the ß-adrenoceptor agonist properties of the isomers of salbutamol. Br J Pharmacol 1973;48. 144–7..
  11. Boulton DW, Fawcett JP. Enantioselective disposition of salbutamol in man following oral and intravenous administration. Br J Clin Pharmacol 1996;41:35-40.[ISI][Medline] [Order article via Infotrieve]
  12. Walle UK, Pesola GR, Walle T. Stereoselective sulphate conjugation of salbutamol in humans: comparison of hepatic, intestinal and platelet activity. Br J Clin Pharmacol 1993;35:413-418.[ISI][Medline] [Order article via Infotrieve]
  13. Walle T, Walle UK, Thornburg KR, Schey KL. Stereoselective sulfation of albuterol in humans: biosynthesis of the sulfate conjugate by HEP G2 cells. Drug Metab Dispos 1993;21:76-80.[Abstract]
  14. Boulton DW, Fawcett JP. Enantioselective disposition of albuterol in humans [Review]. Clin Rev Allergy Immunol 1996;14:115-138.[ISI][Medline] [Order article via Infotrieve]
  15. Dhand R, Goode M, Reid R, Fink JB, Fahey PJ, Tobin MJ. Preferential pulmonary retention of (S)-albuterol after inhalation of racemic albuterol. Am J Respir Crit Care Med 1999;160:1136-1141.[Abstract/Free Full Text]
  16. Ward JK, Dow J, Dallow N, Eynott P, Milleri S, Ventresca P. Enantiomeric disposition of inhaled, intravenous and oral racemic salbutamol in man—no evidence of enantioselective lung metabolism. Br J Clin Pharmacol 2000;49:15-22.[ISI][Medline] [Order article via Infotrieve]
  17. Ventura R, Segura J, Fitch KD, Morton AR, Bergés R, Jiménez C, Berruezo S. Distinction of inhaled and oral salbutamol by urine analysis using conventional screening procedures for doping control. Ther Drug Monit 2000;22:277-282.[ISI][Medline] [Order article via Infotrieve]
  18. Fitch KD. Management of allergic Olympic athletes. J Allergy Clin Immunol 1984;73:722-727.[ISI][Medline] [Order article via Infotrieve]
  19. . IOC Medical Commission. Minutes of meeting. Nagano, February 1998, Lausanne, Switzerland 1998 IOC Medical Commission Lausanne, Switzerland. .
  20. Boulton DW, Fawcett JP. Estimation of drug concentration in plasma from urinary excretion data: an illustration based on salbutamol [Letter]. Eur J Pharmacol 1998;53:373-374.
  21. Bergés R, Segura J, de la Torre X, Ventura R. Analytical methodology for enantiomers of salbutamol in human urine for application in doping control. J Chromatogr B 1999;723:173-184.
  22. Polettini A, Montagna M, Segura J, de la Torre X. Determination of ß2-agonists in hair by gas chromatography/mass spectrometry. J Mass Spectrom 1996;31:47-54.[ISI][Medline] [Order article via Infotrieve]
  23. Ventura R, González G, Smeyers MT, de la Torre R, Segura J. Screening procedure for ß-adrenergic drugs in sports drug testing by immunological methods. J Anal Toxicol 1998;22:127-134.[ISI][Medline] [Order article via Infotrieve]
  24. Bremner P, Woodman K, Burgess C, Crane J, Purdie G, Pearce N, et al. A comparison of the cardiovascular and metabolic effects of formoterol, salbutamol and fenoterol. Eur Respir J 1993;6:204-210.[Abstract]
  25. . British Thoracic Society. Guidelines for management of asthma in adults, 11: acute severe asthma. Br Med J 1990;301:797-800.
  26. . IOC Medical Commission. Statistics on prohibited substances found in sports drug testing 1998 IOC Medical Commission Lausanne, Switzerland. .
  27. Seinsch I, Sigmund G, Horning S, Donike M. Detection of salbutamol in horse urine. In: Donike M, Geyer H, Gotzmann A, Mareck-Engelke U, eds. Recent advances in doping analysis (6). Köln: Sport und Buch Strauss, Editio Sport, 1995:349–55..
  28. Pesola GR, Walle T. Enantiomeric interaction in the sulfate conjugation of the ß2-agonist drug albuterol by the human liver. Res Commun Chem Pathol Pharmacol 1992;75:125-128.[ISI][Medline] [Order article via Infotrieve]
  29. Morgan DJ. Clinical pharmacokinetics of ß-agonists. Clin Pharmacokinet 1990;18:270-294.[ISI][Medline] [Order article via Infotrieve]
  30. Walle T, Eaton EA, Walle UK, Pesola GR. Stereoselective metabolism of R/S-albuterol in humans. Clin Rev Allergy Immunol 1996;14:101-113.[ISI][Medline] [Order article via Infotrieve]
  31. Massart DL, Vandeginste BGM, Deming SN, Michotte Y, Kaufman L. Chemometrics: a textbook 1988:385pp Elsevier Science Publishers BV New York. .
  32. Morris ME, Levy G. Serum concentrations and renal excretion by normal adults of inorganic sulfate after acetaminophen, ascorbic acid, or sodium sulfate. Clin Pharmacol Ther 1983;33:529-536.[ISI][Medline] [Order article via Infotrieve]
  33. Lin JH, Levy G. Effect of prevention of inorganic sulfate depletion on the pharmacokinetics of acetaminophen in rats. J Pharmacol Exp Ther 1986;239:94-98.[Abstract/Free Full Text]
  34. Lown KS, Benet LZ, Watkins PB. Role of intestinal P-glycoprotein in interpatient variation in the oral bioavailability of cyclosporine. Clin Pharmacol Ther 1997;62:248-260.[ISI][Medline] [Order article via Infotrieve]
  35. Anonymous. Levalbuterol for asthma. Med Lett Drugs Ther 1999;41:1054.
  36. Joyce KB, Jones AE, Scott RJ, Biddlecombe RA, Pleasance S. Determination of salbutamol and its 4-O-sulphate metabolites in biological matrices by chiral liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 1998;12:1899-1910.[ISI][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 Journal of Pharmacy PracticeHome page
E. G. Boyce
Use and Effectiveness of Performance-Enhancing Substances
Journal of Pharmacy Practice, February 1, 2003; 16(1): 22 - 36.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergés, R.
Right arrow Articles by de la Torre, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergés, R.
Right arrow Articles by de la Torre, X.
Related Collections
Right arrow Drug Monitoring and Toxicology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS