Beta₂‐adrenergic agonist salbutamol exhibits enantioselective disposition in skeletal muscle of lean young men following oral administration

Abstract

Salbutamol is a common short‐acting beta₂‐adrenergic agonist used in treatment of asthma and exercise‐induced bronchoconstriction but also possesses anabolic and metabolic actions in skeletal muscle. As a chiral compound, salbutamol is a racemic 1:1 mixture of two enantiomers, (R)‐salbutamol and (S)‐salbutamol, which exhibit divergent pharmacokinetic and pharmacodynamic actions. Despite salbutamol being available for decades, information on the enantioselective disposition of salbutamol enantiomers in human skeletal muscle is absent. In this study, we determined concentrations of (R)‐salbutamol and (S)‐salbutamol by UHPLC–MS/MS in arterial plasma and vastus lateralis muscle samples from 12 lean young men 2½ and 7 h following ingestion of 24 mg oral salbutamol. Mean (range) arterial plasma concentrations were 10‐fold higher (p < 0.001) for (S)‐salbutamol than (R)‐salbutamol, being 33(9–62) and 49(30–84) ng·mL⁻¹ for (S)‐salbutamol and 4 (1‐6) and 4 (2‐5) ng·mL⁻¹ for (R)‐salbutamol 2½ and 7 h following administration, respectively, reflecting faster elimination of the (R)‐enantiomer. Mean (range) muscle concentrations were higher (p < 0.001) for (S)‐salbutamol than (R)‐salbutamol 2½ h (0.17 [0.1–0.26] vs. 0.04 [0.02–0.06]) and 7 h (0.31 [0.21–0.46] vs. 0.06 [0.04–0.12] ng·mg_d.w. ⁻¹) after administration. However, muscle:plasma partition coefficient was two‐fold higher (p < 0.001) for (R)‐salbutamol than (S)‐salbutamol 7 h following administration. These observations demonstrate that oral salbutamol exhibits enantioselective disposition in systemic circulation and muscle favoring the (S)‐enantiomer but with higher relative partitioning of the (R)‐enantiomer in skeletal muscle. Furthermore, the concentration‐time profiles of salbutamol enantiomers are different in skeletal muscle and systemic circulation following oral ingestion. These findings have implications for the application of chiral switch (R)‐salbutamol in doping control.

Oral administration of salbutamol shows enantioselective disposition in human skeletal muscle, favoring the (S)‐enantiomer with higher relative partitioning of the pharmacologically active (R)‐enantiomer in muscle. This is concerning from an anti‐doping point of view because the anabolic properties demonstrated for salbutamol can potentially be amplified with chiral switch (R)‐salbutamol.

1. INTRODUCTION

Salbutamol (USAN albuterol) is one of the most commonly prescribed beta₂‐adrenergic agonists (beta₂‐agonists) in humans to relieve symptoms of asthma or asthma‐related conditions. ¹ , ² , ³ The main desired action of salbutamol in these conditions is to induce bronchodilation via stimulation of smooth muscle beta₂‐adrenergic receptors. Yet, beta₂‐adrenergic receptors are abundant throughout the body, giving rise to several extrapulmonary effects of beta₂‐agonists — even when administered via inhalation. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ Given the high density of beta₂‐adrenergic receptors in skeletal muscle ¹² , ¹³ and the apparent effect of beta₂‐agonists on muscle anabolism, ¹⁰ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ studies have investigated the therapeutic application of beta₂‐agonists as treatment in muscle atrophic conditions. ¹⁴ , ¹⁸ Indeed, a period of treatment with beta₂‐agonist causes muscle hypertrophy and increases muscle strength in humans. ¹⁰ , ¹⁵ , ¹⁶ , ¹⁹ , ²⁰ , ²¹

Salbutamol is a chiral compound, consisting of a racemic (rac‐) 1:1 mixture of (R)‐ and (S)‐enantiomers, essentially non‐superimposable mirror image molecules. In pharmacology, individual enantiomers can differ significantly in their specific structure–activity relationships in interactions with transporters, drug receptors, and metabolic enzymes and should be considered separate molecules, rather than a homogenous mixture. Each salbutamol enantiomer has unique pharmacodynamics and pharmacokinetics. ²² , ²³ , ²⁴ , ²⁵ In the case of salbutamol, the desired pharmacological actions reside with the (R)‐enantiomer, whereas the (S)‐enantiomer is generally considered pharmacodynamically inert, which at best could be considered an “inactive by‐product” of manufacture. ²⁵ Several studies have investigated the enantioselective effects of beta₂‐agonist (R)‐ and (S)‐enantiomers in animals and humans for respiratory research. ²⁶ , ²⁷ , ²⁸ , ²⁹ Given the enantioselective disposition of beta₂‐agonists observed in blood and urine from humans, ²³ , ²⁴ , ²⁶ , ³⁰ , ³¹ , ³² , ³³ it seems plausible that skeletal muscles demonstrate significant enantioselective disposition. In rat muscle, salbutamol was shown to exhibit enantioselective disposition following injection ³⁴ and long‐acting beta₂‐agonist, formoterol, was recently shown to exhibit moderate enantioselective distribution in human skeletal muscle of subjects who inhaled formoterol at therapeutic doses. ³⁵ However, despite the demonstrated effects of salbutamol on muscle hypertrophy and strength, and considerable applications in both treatment of muscle‐wasting disease and prohibited doping applications, ²⁰ , ²¹ , ³⁶ , ³⁷ , ³⁸ no studies have investigated the in vivo enantioselective disposition of salbutamol in skeletal muscle of humans. The enantioselective aspects of salbutamol muscle disposition become even more important, considering the availability of “chiral switch” products containing only (R)‐salbutamol (levalbuterol) in some markets.

Herein, we investigated the concentrations and enantioselective disposition of (R)‐ and (S)‐salbutamol in plasma and skeletal muscle of healthy lean young men following oral ingestion of salbutamol using UHPLC–MS/MS (ultra‐high‐performance liquid chromatography–tandem mass spectrometry). Furthermore, we examined the muscle:plasma partition coefficient of each salbutamol enantiomer.

2. MATERIALS AND METHODS

2.1. Volunteer study

Stored arterial plasma and vastus lateralis muscle biopsies sampled from 12 healthy lean young men during a clinical trial ¹⁷ were used for the present study. Subjects were 19–32 years, 172–192 cm in height, had a body mass of 62–89 kg (7–24% body fat), and a leg lean mass of 15–26 kg. Subjects had no history of asthma and were beta₂‐agonist naive. Subjects were informed about potential risks and discomforts related to the study, and each subject gave written and oral informed consent before inclusion. The study was approved by the Committee on Health Research Ethics of the Capital Region of Denmark (H‐1‐2012‐119) and performed in accordance with the 2013 Declaration of Helsinki.

2.2. Sample collection

Subjects met in the morning after an overnight fast and ingested an oral dose of 24 mg salbutamol (Ventolin, GlaxoSmithKline, London, UK) together with a light meal consisting of white bread with jam (energy: 369 kcal; protein: 12 g; carbohydrate: 67 g; fat: 3 g) and 400 mL water. The dose of salbutamol was chosen based on studies showing enhancing effects of oral salbutamol treatment on muscle strength and lean body mass. ¹⁸ , ¹⁹ , ³⁸ Approximately 1½ h after administration, subjects performed eight sets of 12 repetitions of knee‐extensor exercise at an intensity corresponding to 12 repetition maximum (75 ± 11 kg) (mean ± SD) with 2 min of recovery between each set as previously described. ¹⁷ After the exercise, subjects remained inactive in a supine position for 5 h. Arterial blood samples (4 mL) and muscle biopsies were collected ½ and 5 h into recovery from exercise, being equivalent to 2½ and 7 h after administration of salbutamol. The blood sample was drawn in a gel‐free lithium heparin tube (BD vacutainer, NJ, USA) from a catheter in the brachial artery and stood in room temperature for 15 min before being spun at 4000 rpm for 15 min after which plasma was collected and stored at −80°C until analysis. Muscle biopsies were sampled from the vastus lateralis using a 4‐mm Bergström needle with suction. ³⁹ Before sampling, an incision was made through the skin and fascia at the belly of the vastus lateralis muscle during local anesthesia with lidocaine (2 mL lidocaine without epinephrine, Xylocaine® 20 mg·mL⁻¹, AstraZeneca, Cambridge, UK). Muscle biopsy specimens were snap‐frozen in liquid nitrogen and stored at −80°C until analysis.

2.3. Analysis of (R)‐ and (S)‐salbutamol enantiomer levels in plasma and muscle

Plasma and muscle biopsy samples were couriered from University of Copenhagen, Denmark to the University of Tasmania, Australia, under dry ice temperature‐controlled conditions, where the chemical analyses were undertaken. Enantioselective salbutamol analyses followed on from our previous beta₂‐agonist work. ²⁶ , ³⁰ , ³⁴ In brief, freeze‐dried muscle biopsy specimens (approximately 2 mg dry weight) were dissected free of blood, fat, and connective tissue under a microscope using mini scalpels and weighed. The finely dissected tissue was added to a 2‐mL polypropylene tube with 500 μL of dilute ammonia solution at pH ~ 8.5. Then, 1 ng of rac‐salbutamol‐d3 (Toronto Research Chemicals, Toronto, Canada) standard in 10 μL of solution was added. The transfer was completed with 1 mL of ethyl acetate. The tube was then securely capped and vortex mixed for 3 min followed by centrifugation at 12,000 g for 3 min to separate phases. The upper organic phase was transferred into a 2‐mL glass vial, and the solvent was evaporated under a stream of nitrogen gas. The extract was reconstituted in 60 μL methanol and then transferred into a 150 μL glass insert which was placed inside the glass vial and capped, to allow UHPLC–MS/MS analysis. For plasma, 250 μL aliquots of plasma were transferred into a 2‐mL polypropylene tube to which were added 10 ng of rac‐salbutamol‐d3 standard in 10 μL of solution (1 ng·μL⁻¹), dilute ammonia solution to a final pH ~ 8.5 and 1.5 mL ethyl acetate. Extraction of salbutamol and preparation for UHPLC–MS were performed in a similar way as for the muscle tissue samples.

Two sets of calibration standards were prepared using drug‐free muscle tissue and drug‐free plasma, respectively. Drug‐free muscle tissue was spiked with known amounts of rac‐salbutamol (European Pharmacopoeia reference standard from Sigma/Aldrich, Sydney, Australia) equivalent to 0, 0.025, 0.10, 0.25, 0.50, 2.5, and 5.0 ng of each enantiomer and extracted in the same manner as the respective experimental samples. Similarly, drug‐free plasma was spiked with known amounts of rac‐salbutamol equivalent to 0, 1.0, 5.0, 10.0, 20.0, and 100.0 ng·mL⁻¹ of each enantiomer.

All analyses were undertaken using a Waters Acquity® H‐class UHPLC system (Waters Corporation, Milford, MA). Chromatography was performed using an Astec® CHIROBIOTIC™ T chiral column (4.6 · 250 mm · 5 μm particles) (Sigma‐Aldrich). The UHPLC was coupled to a Waters Xevo® triple quadrupole mass spectrometer (Waters Corporation). Analyses were undertaken using multiple reaction monitoring (MRM) in positive electrospray ionization mode.

The UHPLC was operated with a mobile phase consisting of 100% methanol with 0.5% acetic acid and 0.1% ammonium hydroxide. Elution was isocratic for 12 min. The flow rate was 0.8 mL·min⁻¹, and the column was held at 30°C. Injection volume was 40 μL. Electrospray ionization was performed with a capillary voltage of 2.8 kV, a cone voltage of 22 V and individual collision energies for each MRM transition, as described below. The desolvation temperature was 450°C, nebulizing gas was nitrogen at 950 L·h⁻¹, and cone gas was nitrogen at 50 L·h⁻¹. MRM transitions monitored for (R)‐ and (S)‐salbutamol were 240 to 222 m/z, (collision energy 11 V), 240 to 166 m/z (collision energy 14 V), and 240 to 148 m/z (collision energy 18 V), and MRM transitions monitored for (R)‐ and (S)‐salbutamol‐d3 were 243 to 225 m/z (collision energy 11 V), 243 to 169 m/z (collision energy 14 V), and 240 to 151 m/z (collision energy 18 V). Dwell time per channel was 50 ms.

The signal‐to‐noise ratio (S/N) determined for the 0.025 ng muscle calibration standard was used to estimate the lower limit of quantification (LLoQ) in muscle (where S/N = 10) for each enantiomer, by extrapolation. After division by the average biopsy mass, LLoQ was reported as ng·mg⁻¹ dry weight (dw). Accuracy and precision were estimated across the calibration range 0.25–5.0 ng·mg⁻¹ _d.w. for muscle; each n = 4. In a similar manner, the LLoQ in plasma for each enantiomer was determined based on the S/N ratio determined for the 1.0‐ng·mL⁻¹ plasma standard. Accuracy and precision were estimated across the range 5.0–100.0 ng·mL⁻¹ for plasma; each n = 5. For muscle:plasma partition coefficient (K _p ) determinations, 1 ng·mL⁻¹ (plasma) was considered equivalent to 1 ng·g⁻¹ (muscle). Thus, the coefficient was calculated as the muscle (in ng·g dw⁻¹) to plasma (in ng·mL⁻¹) ratio.

Recovery from plasma and muscle was determined at three concentrations in each tissue. Samples were spiked with analyte and together with matched drug‐free tissue were extracted and processed as described previously, but without deuterated internal standard. The drug‐free extracts were then spiked with the same amount of analyte (post extraction) that was used to spike the samples that underwent extraction (thus representing 100% theoretical recovery). The same amount of internal standard was then added to all samples for quantitation before instrumental analysis as described previously. Recovery was estimated from the proportion of rac‐salbutamol post extraction versus rac‐salbutamol from the sample representing 100% theoretical recovery.

2.4. Statistics

Statistical analyses were performed in SPSS version 28 (IBM, Armonk, NY, USA). Data were tested for normality using the Shapiro‐Wilks test and Q‐Q plots. For parametric variables, statistical differences were estimated using a linear mixed model for a repeated measures design with enantiomer (S‐ and R‐salbutamol) and time (2½ and 7 h following administration) as the fixed factors and subjects as a random factor. For non‐parametric variables, differences were estimated using a related‐samples Friedman's Two‐way ANOVA by ranks, which was followed by a Wilcoxon signed rank test to test for differences within specific pairs. Parametric data are presented as mean and standard deviations (SD), unless otherwise stated, whereas non‐parametric data are presented as median and range.

3. RESULTS

3.1. Enantioselective UHPLC–MS/MS assay performance

All matrices met acceptance criteria for accuracy (<15% deviation), precision (<15%RSD), recovery (>20%) in plasma and muscle, and estimate of linearity (r ² > 0.999). For muscle biopsy determinations, the LLoQ was influenced by the mass of tissue obtained, with lower concentrations achieved with larger mass biopsies. Mean muscle biopsy sample mass (SD) for dry weight (dw) was 2.31 (0.38) mg resulting in an LLoQ estimate of 1.9 pg·mg⁻¹and 2.5 pg·mg⁻¹ for (R)‐ and (S)‐salbutamol, respectively. For plasma, the LLoQ was 96 and 94 pg·mL⁻¹ for (R)‐ and (S)‐salbutamol, respectively. In muscle, accuracy was −3.0% and −3.5% for (R)‐ and (S)‐salbutamol, respectively, and precision was 4.2% and 2.3% RSD for (R)‐ and (S)‐salbutamol, respectively. In plasma, accuracy was −3.6% and −2.6% for (R)‐ and (S)‐salbutamol, respectively, and precision was 7.1%RSD and 5.0%RSD for (R)‐ and (S)‐salbutamol, respectively. Example chromatograms of a muscle sample are shown in Figure 1.

FIGURE 1.

UHPLC–MS/MS chromatogram from muscle sample equivalent to 0.07 ng·mg⁻¹ and 0.43 ng·mg⁻¹ (dry weight) salbutamol enantiomers for (R)‐salbutamol and (S)‐salbutamol respectively. (a) Salbutamol deuterated standard. (b) rac‐salbutamol. UHPLC–MS/MS, ultra‐high‐performance liquid chromatography–tandem mass spectrometry.

3.2. (R)‐ and (S)‐salbutamol enantiomer disposition in plasma and skeletal muscle

Enantiomer levels of (R)‐ and (S)‐salbutamol in plasma and in muscle biopsies of the vastus lateralis 2½ and 7 h after ingestion of salbutamol are presented in Figures 2 and 3). Mean arterial plasma concentrations were around 10‐fold higher (p < 0.001) for (S)‐salbutamol than (R)‐salbutamol (Figure 2(a + b)). Arterial plasma concentrations of (S)‐salbutamol rose (p < 0.01) from 2½ to 7 h following administration, whereas no changes were observed in the similar period for (R)‐salbutamol.

FIGURE 2.

Plasma concentrations of (R)‐ and (S)‐salbutamol 2½ and 7 h after ingestion of 24 mg oral salbutamol. (a) (S)‐salbutamol. (b) (R)‐salbutamol. (c) Enantiomer ratios. *Different from 2½ h (p < 0.05).

FIGURE 3.

Muscle content of (R)‐ and (S)‐salbutamol 2½ and 7 h after ingestion of 24 mg oral salbutamol. (a) (S)‐salbutamol. (b) (R)‐salbutamol. (c) Enantiomer ratios. *Different from 2½ h (p < 0.05).

In vastus lateralis muscle biopsies, concentrations of (S)‐salbutamol were five‐fold higher (p < 0.01) than the (R)‐salbutamol concentrations (Figure 3(a + b)). A two‐fold increase in the muscle concentrations was observed for both salbutamol enantiomers from 2½ to 7 h following administration. The estimated amount of salbutamol enantiomers deposited in vastus lateralis muscle was 1.7 ± 0.4 and 0.4 ± 0.1 mg for (S)‐ and (R)‐salbutamol (p < 0.01), respectively, 2½ h after administration, increasing (time main effect: p < 0.01) to 3.0 ± 1.4 and 0.6 ± 0.4 mg (p < 0.01) 7 h after administration.

The (R):(S) concentration ratio declined (p < 0.01) in arterial plasma from 2½ to 7 h after administration with a trend for an opposite pattern in muscle (Figures 2c and 3c). At both sampling times, the (R):(S)‐concentration ratio was higher (p < 0.01) in muscle than in arterial plasma.

3.3. Muscle:plasma partition coefficient

The median (range) muscle:plasma partition coefficient (K _p ) was not different (p = 0.241) between the salbutamol enantiomers 2½ h following administration; 1.5 (0.8–3.1) for (S)‐salbutamol and 1.8 (1.3–4.7) for (R)‐salbutamol, whereas K _p was higher (p = 0.003) for (R)‐salbutamol than (S)‐salbutamol 7 h following administration; 3.69 (2.4–14.1) for (R)‐salbutamol and 0.99 (0.5–3.9) for (S)‐salbutamol (Figure 4(a + b)). K _p increased from 2½ to 7 h following administration for (R)‐salbutamol (p = 0.045) but not for (S)‐salbutamol (p = 0.358) (Figure 4(a + b)).

FIGURE 4.

Muscle:plasma (K _p ) partition coefficient of (R)‐salbutamol (a) and (S)‐salbutamol (b) 2½ and 7 h after ingestion of 24 mg oral salbutamol. Content of muscle salbutamol was divided by 4 under the assumption of 4‐fold greater mass of wet weight tissue than dry weight. Box plots are median with interquartile ranges and whiskers are minimum to maximum. *Different from 2½ h (p < 0.05).

4. DISCUSSION

The most important observations of the present study were that salbutamol exhibited enantioselective disposition in both the systemic circulation and in skeletal muscle of lean young males who ingested salbutamol at high oral doses.

We observed that skeletal muscle is a major site of (R)‐ and (S)‐salbutamol disposition following ingestion of an oral dose of 24 mg salbutamol. The (S)‐enantiomer was present at approximately 2‐fold higher muscle concentrations than the (R)‐enantiomer. This is likely reflective of greater arterial availability of (S)‐salbutamol, approximately 10‐fold higher than (R)‐salbutamol, and is consistent with previous findings regarding systemic clearance being 3 times higher for (R)‐ than (S)‐salbutamol as also reflected by reported half‐lives in the order of 2.5 h for (R)‐salbutamol versus 5 h for (S)‐salbutamol. ²²

Despite the higher (S)‐salbutamol concentrations than (R)‐salbutamol concentrations in muscle, we observed that the muscle:arterial plasma partition coefficient K _p was two‐fold higher for the (R)‐enantiomer than for the (S)‐enantiomer 7 h following oral ingestion. This is consistent with the K _p observed for beta₂‐agonist formoterol in humans ³⁵ and for salbutamol in rodents, ³⁴ and indicates that beta₂‐agonists exhibit enantioselective distribution in skeletal muscle in favor of the (R)‐enantiomers. It can thus be expected that injection of salbutamol, and even inhalation of salbutamol, exhibiting less pronounced enantioselective disposition in the systemic circulation, ²² , ²³ , ²⁵ would give rise to proportionally even higher (R)‐enantiomer levels in skeletal muscle than (S)‐enantiomer levels. Indeed, in rodents injected with salbutamol, the (R)‐enantiomer was present at much higher levels than the (S)‐enantiomer in abdominal muscles. ³⁴ Likewise, formoterol concentrations of the (R,R)‐enantiomer were shown to be slightly higher than the (S,S)‐enantiomer in human skeletal muscle following inhalation of racemic formoterol therapeutic doses. ³⁵

The substantial enantioselective disposition of salbutamol enantiomers in the circulation following oral ingestion was expected and consistent with previous reports. ²² , ²³ Ward et al. ²² notably observed that the route of administration was a determinant of the extent of enantioselective disposition in venous plasma. As in the present study, the authors observed that venous plasma concentrations of the (S)‐enantiomer were approximately 10‐fold higher than the (R)‐enantiomer following oral ingestion of salbutamol. ²² However, when administered via inhalation or when injected, the degree of enantioselective disposition was less extensive. High levels of the (S)‐enantiomer relative to the (R)‐enantiomer following oral ingestion are related to a greater rate of absorption of the (S)‐enantiomer from the gastro‐intestinal system and/or a faster rate of gastro‐intestinal and hepatic metabolism of the (R)‐enantiomer. ²² In addition, the higher muscle:plasma partition coefficient for the (R)‐enantiomer, as observed in the present study, would contribute to a faster rate of clearance of the (R)‐enantiomer from the systemic circulation into skeletal muscle relative to the (S)‐enantiomer. Finally, enantioselective elimination from the systemic compartment may be contributing to the higher concentrations of the (S)‐enantiomer, as higher renal clearance has been reported for the (R)‐enantiomer of salbutamol. ²²

While the time course for (S):(R) salbutamol uptake in muscle shows a consistent increase over the time course of 2½ and 7 h after administration, we are unable to speak to the elimination from the muscle compartment. For example, while salbutamol is largely cleared from the systemic compartment within ~24 h after administration, it is worth investigating how long the drug remains in the muscle compartment and whether the duration of action on muscle tissue persists for longer than the presence in the systemic compartment. In addition, salbutamol as a therapeutic inhaled dose may offer a more applicable setting as it is considered the first‐line route of administration for asthma treatment. It should be expected that some muscle disposition occurs, as even therapeutic dose administration reaches the systemic circulation and elicits muscle hypertrophy during chronic treatment, ¹⁰ , ⁴⁰ but the contribution of intramuscular disposition of beta₂‐agonist in this response is not clear. Intramuscular disposition could be a contributing mechanism explaining the difference in magnitude of the physiological response observed between different beta₂‐agonists. ⁴¹

Taken together, the present study shows that salbutamol, when ingested orally, exhibits enantioselective disposition in the systemic circulation and skeletal muscle in favor of the (S)‐enantiomer but with a higher relative partitioning of the pharmacologically active (R)‐enantiomer in muscle of lean young men. This is concerning from an anti‐doping point of view because the anabolic properties demonstrated for salbutamol can potentially be amplified with chiral switch (R)‐salbutamol. Furthermore, our data suggest that the pharmacokinetic concentration‐time profiles of the salbutamol enantiomers are different in skeletal muscle compared to the systemic circulation following oral ingestion. In conclusion, the distinct pharmacokinetic profiles of the salbutamol enantiomers suggests that careful consideration must be given to the use of chiral switch products consisting of enantiopure (R)‐salbutamol to avoid performance enhancing effects and to ensure fair doping control.

AUTHOR CONTRIBUTIONS

Participated in research design: Hostrup and Jacobson; conducted experiments: Hostrup, Eibye, Jessen, Narkowicz; contributed new reagents or analytic tools: Nichols, Narkowicz, Jacobson; performed data analysis: Hostrup, Eibye, Jessen, Nichols, Jacobson; wrote or contributed to the writing of the manuscript: Hostrup, Eibye, Jessen, Narkowicz, Nichols, Jacobson. All authors approved the final version of the manuscript.

Hostrup M, Jacobson GA, Eibye K, Narkowicz CK, Nichols DS, Jessen S. Beta₂‐adrenergic agonist salbutamol exhibits enantioselective disposition in skeletal muscle of lean young men following oral administration. Drug Test Anal. 2025;17(6):842‐849. doi: 10.1002/dta.3787