The Clinical Practice and Best Aerosol Delivery Location in Intubated and Mechanically Ventilated Patients: A Randomized Clinical Trial.

This randomized clinical trial (RCT) is aimed at exploring the best nebulizer position for aerosol delivery within the mechanical ventilation (MV) circuitry. This study enrolled 75 intubated and MV patients with respiratory failure and randomly divided them into three groups. The nebulizer position of patients in group A was between the tracheal tube and Y-piece. For group B, the nebulizer was placed at the inspiratory limb near the ventilator water cup (80 cm away from the Y-piece). For group C, the nebulizer was placed between the ventilator inlet and the heated humidifier. An indirect competitive enzyme-linked immunosorbent assay (ELISA) was used to measure salbutamol drug concentrations in serum and urine. The serum and urine salbutamol concentrations of the three groups were the highest in group B, followed by group C, and the lowest in group A. Serum and urine salbutamol concentrations significantly differed among the three groups (P < 0.05). It was found that the drug was statistically significant between group differences for groups B and A (P = 0.001; P = 0.002, respectively) for both serum and urine salbutamol concentrations. There were no significant differences observed among the other groups. It was found that the drug concentrations were the highest when the nebulizer was placed 80 cm away from the Y-piece, while the location between the tracheal tube and the Y-piece with the higher frequency of nebulizer placement was the location with the lowest drug concentration.

Rapid Detection of Ractopamine and Salbutamol in Swine Urine by Immunochromatography Based on Selenium Nanoparticles.

PURPOSE: The purpose of this study was to establish a lateral flow immunoassay using selenium nanoparticles (Se-NPs) as a probe to detect ractopamine (RAC) and salbutamol (SAL) in swine urine. METHODS: SDS and PEG were used as templates to prepare Se-NPs; anti-RAC monoclonal antibodies or anti-SAL monoclonal antibodies were labelled with Se-NPs; and rapid detection kits were prepared. The sensitivity, specificity, and stability were measured, and actual samples were analysed. RESULTS: The Se-NPs were spherical with a diameter of 40.63 ± 5.91 nm, and were conjugated successfully with an anti-RAC antibody to give a total diameter of 82.33 ± 17.91 nm. The detection limit of a RAC kit in swine urine was 1 ng/mL, and that of a SAL kit was 3 ng/mL. Both procedures could be completed within 5 minutes. No cross-reaction occurred with clenbuterol, bambuterol and phenylethanolamine A. Samples were tested consistently across different batches of kits for swine urine. The results of the kits were identical to those of actual clinical samples analysed by ELISA, and the coincidence rate was 100%. CONCLUSION: The assay kit does not require any special device for reading the results, and the readout is a simple colour change that can be evaluated with the naked eye. It is easy to operate, sensitive, specific, and stable This kit is suitable for the rapid and real-time detection of RAC and SAL residues in swine urine samples. CLINICAL TRIAL REGISTRATION: Swine urines samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University.

Comparative pharmacokinetics of salbutamol inhaled from a pressurized metered dose inhaler either alone or connected to a newly enhanced spacer design.

BACKGROUND: Coordination between actuation of a pressurized metered dose inhaler (pMDI) and inhalation is a critical manoeuvre that many patients fail to perform correctly. pMDIs connected to spacers do not require hand-lung coordination. This study evaluated the relative lung and systemic bioavailability and oropharyngeal deposition of salbutamol post-inhalation from Ventolin® Evohaler® (GlaxoSmithKline) either alone following verbal inhaler technique counselling (VC) or connected to a newly improved Able Spacer® (AS). METHODS: In a two-period, randomized crossover study, 16 healthy adults inhaled 2 × 100 µg salbutamol puffs (1 min gap) from Ventolin using VC or AS. Immediately after each puff inhalation, each subject gargled with 20 mL water for oropharyngeal deposition (OD) determination. Urine samples were collected 0.5 h (pre-) and 0.5, 1.0 and 2.0 h post-inhalation. Urine was then pooled 2-24 h post-inhalation. The relative lung bioavailability (0-0.5 h urinary salbutamol excretion - USAL0.5) and systemic bioavailability (0-24 h urinary excretion of salbutamol and its metabolite - USALMET24) were determined. A one week washout separated VC and AS use. RESULTS: The mean (SD) USAL0.5 of VC and AS was 5.36 (4.48) and 12.80 (10.83) µg, respectively. The mean (SD) OD was 11.35 (3.37) and 0.48 (0.30) µg, respectively. VC and AS were significantly different in USAL0.5 and OD (p<0.001). USALMET24 was comparable (p>0.05). CONCLUSIONS: Compared with VC, AS doubled the inhaled salbutamol lung dose and minimised its precipitation in the oral cavity. The results suggest this inhalation aid can add therapeutic and safety benefits particularly in patients with continued pMDI technique issues despite repeated VC.

Novel paper-based colorimetric immunoassay (PCI) for sensitive and specific detection of salbutamol residues in flesh of swine and urine using Ag3 PO4 /Ag nanocomposite as label.

Salbutamol (SAL) can cause potential hazards to human health and its use as a growth promoter in meat-producing animals is illegal. This work reports a novel approach for competitive paper-based colorimetric immunoassay (PCI) using the Ag3 PO4 /Ag nanocomposite as label for sensitive and specific determination of SAL in flesh of swine and urine. The Ag3 PO4 /Ag nanocomposite was synthesized by a one-step chemical bath method, which could instantly oxidize a chromogenic substrate for the color development under acidic conditions without the participation of H2 O2 . This approach provides high affinity between the Ag3 PO4 /Ag nanocomposite and the substrate (with the Michaelis-Menten constant of 0.44 mM). In addition, the fabrication process of the PCI was simple and cost-effective. Particularly, the novel PCI also exhibits simplicity and cost-effectiveness of the fabrication process through a simple wax screen-printing, which requires inexpensive equipment and material including a screen, wax, a squeegee, and a hair dryer. Under optimal conditions, the competitive PCI exhibited a linearity range of 0.025 to 1.00 µg/L. The developed approach offers advantages over the conventional ELISA for the purpose of routine use because it requires a shorter incubation time (<1 hr), significantly small volumes of reagents and samples (<100 µL each), and an inexpensive consumer-grade digital camera coupled with a simple gray-scale transformation of the RGB (Red Green Blue) color image for the purpose of quantification of the detection. PRACTICAL APPLICATION: Salbutamol (SAL) can cause potential hazards to human health and the use of which as growth promoter in meat-producing animals is illegal. This work introduces a novel approach for competitive immunoassay on paper-based colorimetric immunoassay using the Ag3 PO4 /Ag nanocomposite as the label (instead of using natural enzyme) for low-cost, sensitive, and specific determination of SAL residues at low level in flesh of swine and urine samples. The proposed approach offers advantages over the conventional ELISA for the purpose of routine use.

Simultaneous Detection of Multiple ß-Adrenergic Agonists with 2-Directional Lateral Flow Strip Platform.

Clenbuterol (CL), salbutamol (SAL) and ractopamine (RAC) are the three common ß-adrenergic agonists, which are the main hazards in food safety and affect human health through the food chain. A convenient and efficient method is urgently required to perform on-site detection of multiple ß-adrenergic agonists to avoid frequent poisoning incidents. In this paper, a 2-directional lateral flow strip technique (2-directional LFS) is developed for rapid and simultaneous detection of CL, SAL and RAC with single sampling. Compared to the conventional lateral flow strip, this 2-directional LFS technique can realize simultaneous detection of three or more target analytes without any change of intrinsic simplicity of LFS. Furthermore, this 2-directional LFS can effectively avoid the potential intrinsic cross-reactivity among the reagents to analogues. Under the optimized conditions, CL, SAL and RAC were all successfully determined with satisfactory results in both buffer and urine samples with the detection limit as low as 0.5 ng/mL. This 2-directional LFS technique can revolutionize the commercial single-analyte LFS products and can effectively widen the applications of the classic LFS in various fields.

The use of hair as a long-term indicator of low-dose ß2 agonist treatments in cattle: Implications for growth-promoting purposes monitoring.

The objective of this study was to assess the feasibility of using hair as a long-term indicator of cocktail (low-dose ß2 agonists) treatments in cattle. Six male Simmental cattle were treated with a mixture of low-dose clenbuterol, ractopamine, and salbutamol at dosages of 5.3, 223.3, and 50.0 µg/kg, respectively. The trial lasted for 112 days and included 28 days of treatment and 84 days of withdrawal. Plasma and urine samples taken during the treatment period contained the highest residues, with maximum concentrations of clenbuterol, ractopamine, and salbutamol in plasma of 1.49 ng/mL (Day 21), 43.78 (Day 14) ng/mL, and 8.07 ng/mL (Day 7), respectively, and in urine of 62.40 ng/mL (Day 28), 3995.77 ng/mL (Day 28), and 503.72 ng/mL (Day 1), respectively. On day 42 of withdrawal, the residues of all three ß2 agonists in plasma were below the limit of quantification (LOQ; 0.3 ng/mL for clenbuterol, and 0.5 ng/mL for ractopamine and salbutamol), and in urine samples were below or near the LOQ (the highest being ractopamine at 1.10 ng/mL). The highest concentrations of clenbuterol, ractopamine, and salbutamol in hair were 88.36, 1351.92, and 100.58 ng/g, respectively, on day 14 of withdrawal; and the residues were long-lasting, with 7.64, 28.55, and 8.77 ng/g, respectively, on day 84 of withdrawal. The results of this study demonstrate that hair could be utilized as a long-term indicator of the use of a combination of low-dose ß2 agonists in cattle, which could have implications for growth-promoting purposes monitoring.

Effects of Fill Volume and Humidification on Aerosol Delivery During Single-Limb Noninvasive Ventilation.

BACKGROUND: The aim of this work was to determine the effect of fill volume and humidification change on aerosol delivery during single-limb noninvasive ventilation (NIV). METHODS: Four groups were recruited, each consisting of 12 subjects (6 females) with COPD receiving NIV. Groups 1 and 3 received inhaled salbutamol with a vibrating mesh nebulizer, and Groups 2 and 4 received inhaled salbutamol with a jet nebulizer. The in vivo study was carried out on days 1 and 3. In groups 1 and 2, 2 fill-volumes were delivered to each subject; 1 mL 5,000 µg/mL salbutamol respirable solution used as it is or diluted to a total of 2 mL using normal saline. In groups 3 and 4, 1 mL 5,000 µg/mL salbutamol respirable solution diluted to 2 mL total volume using normal saline was delivered to each subject with and without humidification. Unchanged salbutamol in urine at 30 min (USAL0.5) and in pooled urine at 24 h (USAL24) was determined. On day 2, the ex vivo study was carried out on subjects using the same experimental setting with a filter placed proximal to their face mask for collection of total inhaled dose of salbutamol (aerosol emitted). RESULTS: The vibrating mesh nebulizer delivered higher USAL0.5, USAL24, and aerosol emitted compared to the jet nebulizer at all fill volumes and humidification conditions (P < .001). Increasing fill volume from 1 mL to 2 mL resulted in a significant increase in USAL0.5, USAL24, and aerosol emitted from the jet nebulizer (P < .05) with an insignificant effect on the vibrating mesh nebulizer. A 2-mL fill volume with the jet nebulizer delivered USAL24 and aerosol emitted comparable to those of 1 mL with the vibrating mesh nebulizer with significantly longer nebulization times (P < .001). Humidification had an insignificant effect on aerosol delivery. CONCLUSIONS: Increasing the fill volume of a jet nebulizer is essential to increase the amount of inhaled medication reaching a subject. In contrast, there is no need to increase fill volumes when using a vibrating mesh nebulizer. There is no need to switch off the humidifier while delivering aerosol through a single-limb NIV circuit.

Performance of Large Spacer Versus Nebulizer T-Piece in Single-Limb Noninvasive Ventilation.

BACKGROUND: Predosing patients with COPD with salbutamol by using a pressurized metered-dose-inhaler (pMDI) as a bronchodilator was hypothesized to improve the distribution of the subsequent nebulized dose. This study determined the effect of a pMDI preliminary bronchodilator dose on the aerosol delivered by a mesh nebulizer during single-limb noninvasive ventilation. METHODS: Twelve subjects with COPD who received noninvasive ventilation were enrolled in a randomized, open-label, urinary pharmacokinetic study. A bi-level ventilator with a dry single-limb circuit and the fixed expiratory port was set in the spontaneous mode, with initial inspiratory and expiratory pressures of 20 and 5 cm H2O respectively, a 1:3 inspiratory-expiratory ratio, and 15 breaths/min. Salbutamol was administered via a mesh nebulizer with a large spacer or T-piece placed between the fixed-orifice expiratory valve and the oronasal mask. In vivo dosing methods were randomized for days 1, 3, and 5 of the study. On each day, a 1-mL respirable solution that contained 5,000 µg salbutamol was nebulized by using a mesh nebulizer with 3 setting: (1) T-piece, (2) large spacer, and (3) large spacer plus pMDI. Only with the large spacer plus pMDI setting, 2 pMDI doses, which contained 100 µg salbutamol each, were actuated before nebulization. Urine samples were collected at 0.5 h (as an index of pulmonary bioavailability) and pooled up to 24 h after dosing (as an index of systemic absorption). On day 2, ex vivo studies were performed for the 3 setting with salbutamol collected onto filters placed before the mask. The drug was eluted from the filters and analyzed to determine the inhaled dose. RESULTS: A large spacer plus pMDI showed a trend to deliver a higher fraction (percentage of nominal dose) of both ex vivo filters and 0.5-h urinary salbutamol. The 0.5-h urinary salbutamol excreted with a large spacer plus pMDI (1.99%) was larger than with the T-piece (1.73%) and large spacer (1.78%). This trend did not extend to the 24-h levels, in which bioavailability with the large spacer plus pMDI (49.9%) was lower than with the T-piece (52.8%) and with the large spacer (54.3%). However, no differences were significant. CONCLUSIONS: The T-piece and large spacer were equally efficient for salbutamol delivery from the mesh nebulizer in patients with COPD and on single-limb noninvasive ventilation. Adding a preliminary bronchodilator dose by pMDI prenebulization showed a trend toward greater pulmonary bioavailability of nebulized salbutamol and may be worth considering to maximize delivery of salbutamol to patients who are severely ill.

Effect of a Nebulizer Holding Chamber on Aerosol Delivery.

BACKGROUND: A new holding chamber was designed to be used with the Aerogen Solo nebulizer to increase the aerosol emitted that reach the patient. The aim of this study was to evaluate the efficacy of this holding chamber with the nebulizer and determine its usability with other nebulizers. METHODS: The study was divided into 2 parts. In the first part, aerosol emitted of 1 mL respirable solution (nominal dose of 5000 µg salbutamol), delivered by using the mesh nebulizer, Pro nebulizer, and jet nebulizer, connected to a T-piece or a holding chamber, was determined by using a breathing simulator set to provide a tidal volume of 500 mL, frequency of 15 breaths/min, and the inspiratory-expiratory ratio of 1:1 for adults as the quiet breathing pattern. Aerodynamic particle size characterizations were determined by using a cooled cascade impactor at an inhalation flow of 15 L/min. In the second part of the study, 12 healthy nonsmoking subjects (6 females) >18 y, with an FEV1 > 90% were enrolled. Inhaled aerosol of 1 mL respirable solution (5,000 µg salbutamol) was delivered through the mesh nebulizer-holding chamber and an mesh nebulizer-T-piece using normal tidal breathing. The subjects provided urine samples 30 min after dosing and cumulatively collected their urine for 24 h. The samples were analyzed for salbutamol content. RESULTS: The holding chamber significantly increased aerosol emitted by the 3 nebulizers compared with the T-piece (P < .01) and relatively decreased the mass median aerodynamic diameter but with no significant difference. The mesh nebulizer-holding chamber resulted in significantly higher aerosol emitted compared with any other delivery method tested (P < .01). The mesh nebulizer-holding chamber resulted in higher urine samples 30 min after dosing (as an index of lung deposition) and cumulatively collected urine for 24 h (as an index of systemic absorption) compared with the nebulizer-T-piece (P < .05). CONCLUSIONS: The use of the holding chamber with a jet nebulizer, Pro nebulizer, and the Solo nebulizer significantly increased the aerosol delivery. The Solo nebulizer-holding chamber had the highest aerosol emitted compared with all nebulizer-adapter combinations and higher urine samples 30 min after dosing and cumulatively collected urine for 24 h compared with the nebulizer-T-piece.

Isolation and characterization of a novel anti-salbutamol chicken scFv for human doping urinalysis.

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant ß2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two ß2-agonists were; IC50 salbutamol = ∼0.310 µg/ml, IC50 clenbuterol = ∼0.076 µg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 µg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 µg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.

Futility of current urine salbutamol doping control.

AIMS: Salbutamol is used in the management of obstructive bronchospasm, including that of some elite athletes. It is claimed that high salbutamol (oral) doses may also have an anabolic effect. Therefore, inhalation of salbutamol is restricted by the World Anti-Doping Agency (WADA) to a maximal daily dose. Urine is tested for violations, but recent cases have resulted in a debate regarding the validity of this approach. It was our aim to determine whether current approaches are sufficiently able to differentiate approved usage from violations. METHODS: We extracted pharmacokinetic parameters from literature for salbutamol and its sulphated metabolite. From these parameters, a semi-physiological pharmacokinetic model of inhaled and orally administered salbutamol was synthesized, validated against literature data, and used to perform clinical trial simulations (n = 1000) of possible urine concentrations over time resulting from WADA-allowed and oral unacceptable dosages. RESULTS: The synthesized model was able to predict the literature data well. Simulations showed a very large range of salbutamol concentrations, with a significant portion of virtual subjects (15.4%) exceeding the WADA threshold limit of 1000 ng ml-1 at 1 h post-dose. CONCLUSIONS: The observed large variability in urine concentrations indicates that determining the administered dose from a single untimed urine sample is not feasible. The current threshold inadvertently leads to incorrect assumptions of violation, whereas many violations will go unnoticed, especially when samples are taken long after drug administration. These issues, combined with the dubious assertion of its anabolic effect, leads us to conclude that the large effort involved in testing should be reconsidered.

Residues of Salbutamol and Identification of Its Metabolites in Beef Cattle.

Salbutamol, a selective ß2-agonist, endangers the safety of animal products because of its illegal use in food animals. In this work, residues of salbutamol and its metabolites were investigated to select appropriate targets and marker residues for monitoring the illegal use of salbutamol. Ten metabolites of salbutamol were identified from plasma, urine, liver, and kidney samples; of these, six were newly identified. There were significant differences (P < 0.01) between the parent (nonconjugated) and total (conjugated + nonconjugated) salbutamol concentrations in plasma, urine, liver, and kidney tissues. Salbutamol residues in urine were relatively higher than those in plasma and other internal tissues during the dosing period and were rapidly eliminated from plasma, heart, spleen, and kidney tissues during the withdrawal time. Total salbutamol was identified as more preferable than parent salbutamol as a marker residue, and urine and eye tissues were found to be more suitable as targets for preslaughter and postslaughter monitoring of the illegal use of salbutamol in beef cattle.

The enigma of inhaled salbutamol and sport: unresolved after 45 years.

During the past 45 years, there have been more changes on the World Anti-Doping Agency's (WADA) Prohibited List (the List) to the status of inhaled salbutamol than any other substance. With 658 athletes, 6.1% of all participating athletes approved to inhale salbutamol at the 2008 Beijing Games, it is one of the medications used most frequently by Olympic athletes. Nevertheless, since the 2008 Games, WADA has made numerous changes to inhaled salbutamol on the List including prohibiting its use, then a year later permitting it without prior notification and recommending a pharmacokinetic study if an athlete exceeds the urinary threshold of 1000 ng/mL. Recently, an elite athlete undertook two pharmacokinetic studies and the results have raised several questions. These include whether WADA should continue to permit nebulized salbutamol as an acceptable method of inhalation and there is some justification for nebulized salbutamol to be prohibited in sport. Another question is whether the modified advisory on salbutamol in the 2017 List appropriately informs athletes of the risks of exceeding the urinary threshold and the recent changes may not inform athletes optimally. Finally, concern is expressed at the persistent failure of WADA to apply a correction down to a specific gravity of 1.020 when an exogenous substance is identified in the urine of a dehydrated athlete. It is recommended that this should be implemented. Copyright © 2017 John Wiley & Sons, Ltd.

Ag/N-doped reduced graphene oxide incorporated with molecularly imprinted polymer: An advanced electrochemical sensing platform for salbutamol determination.

In this work, the metallic silver and non-metallic nitrogen co-doped reduced graphene oxide (Ag-N-RGO) was first synthesized by a simple and cost-effective strategy, and then a molecularly imprinted polymer (MIP) was formed in situ at the surface of the prepared composite via electropolymerization of o-phenylenediamine in the presence of salbutamol as the template molecule. The electrochemical characterizations demonstrate that the bifunctional graphene-based composite shows improved catalytic performance than that of pristine graphene doped with one-component or none. The MIP sensor based on Ag-N-RGO owns high porous surface structure, resulting in the increased current response and enhanced recognition capacity than that of non-imprinted sensor. The outstanding performance of the developed sensor derives from the combined advantages of Ag-N-RGO with effective catalytic property and MIP with excellent selectivity. Under the optimal conditions, the electrochemical response of the developed sensor is linearly proportional to the concentration of salbutamol in the range of 0.03-20.00µmolL-1 with a low detection limit of 7 nmol L-1. The designed sensor has exhibited the multiple advantages such as low cost, simple manufacture, convenient use, excellent selectivity and good reproducibility. Finally, the proposed method has been extended for the determinations of salbutamol in human urine and pork samples, and the satisfactory recoveries between 98.9-105.3% are achieved.

How can we optimise inhaled beta2 agonist dose as 'reliever' medicine for wheezy pre-school children? Study protocol for a randomised controlled trial.

BACKGROUND: Asthma is a common problem in children and, if inadequately controlled, may seriously diminish their quality of life. Inhaled short-acting beta2 agonists such as salbutamol are usually prescribed as 'reliever' medication to help control day-to-day symptoms such as wheeze. As with many medications currently prescribed for younger children (defined as those aged 2 years 6 months to 6 years 11 months), there has been no pre-licensing age-specific pharmacological testing; consequently, the doses currently prescribed (200-1000 µg) may be ineffective or likely to induce unnecessary side effects. We plan to use the interrupter technique to measure airway resistance in this age group, allowing us for the first time to correlate inhaled salbutamol dose with changes in clinical response. We will measure urinary salbutamol levels 30 min after dosing as an estimate of salbutamol doses in the lungs, and also look for genetic polymorphisms linked to poor responses to inhaled salbutamol. METHODS: This is a phase IV, randomised, controlled, observer-blinded, single-centre trial with four parallel groups (based on a sparse sampling approach) and a primary endpoint of the immediate bronchodilator response to salbutamol so that we can determine the most appropriate dose for an individual younger child. Simple randomisation will be used with a 1:1:1:1 allocation. DISCUSSION: The proposed research will exploit simple, non-invasive and inexpensive tests that can mostly be performed in an outpatient setting in order to help develop the evidence for the correct dose of salbutamol in younger children with recurrent wheeze who have been prescribed salbutamol by their doctor. TRIAL REGISTRATION: EudraCT2014-001978-33, ISRCTN15513131. Registered on 8 April 2015.

Beta2-Agonist Doping Control and Optical Isomer Challenges.

The World Anti-Doping Agency (WADA) currently allows therapeutic use of the beta2-agonists salbutamol, formoterol and salmeterol when delivered via inhalation despite some evidence suggesting these anti-asthma drugs may be performance enhancing. Beta2-agonists are usually administered as 50:50 racemic mixtures of two enantiomers (non-superimposable mirror images), one of which demonstrates significant beta2-adrenoceptor-mediated bronchodilation while the other appears to have little or no pharmacological activity. For salbutamol and formoterol, urine thresholds have been adopted to limit supratherapeutic dosing and to discriminate between inhaled (permitted) and oral (prohibited) use. However, chiral switches have led to the availability of enantiopure (active enantiomer only) preparations of salbutamol and formoterol, which effectively doubles their urine thresholds and provides a means for athletes to take supratherapeutic doses for doping purposes. Given the availability of these enantiopure beta2-agonists, the analysis of these drugs using enantioselective assays should now become routine. For salmeterol, there is currently only a therapeutic dose threshold and adoption of a urinary threshold should be a high priority for doping control.

Salbutamol Residues in Plasma, Urine and Hair of Heifers After a Single Dose and Throughout.

The objective of this study was to evaluate the salbutamol residues in the plasma, urine and hair of heifers after a single dose. Three heifers were given a single oral dose of salbutamol hydrochloride (150 µg/kg bodyweight). The salbutamol concentrations were measured in the plasma, urine (before and after hydrolysis with ß-glucuronidase/arylsulfatase) and hair samples with ultra-performance liquid chromatography-tandem mass spectrometry. In the unhydrolyzed samples, the peak concentrations of salbutamol occurred in the plasma and urine at 12 and 8 h after drug administration, respectively, but were below the limit of quantification (LOQ = 0.2 ng/mL) at 48 and 120 h after administration, respectively. However, in the hydrolyzed samples, the salbutamol concentration was 1.1 ng/mL in the plasma 72 h after its administration and 0.7 ng/mL in the urine 168 h after its administration. Thus, the concentrations of salbutamol were significantly higher in the hydrolyzed samples than that in the unhydrolyzed samples (P < 0.01). The concentrations of salbutamol in the black and white hair 24 h after its administration were 1.7 and 1.0 ng/g, respectively. These results indicate that hair may be a target tissue for detecting the misuse of salbutamol after a single dose and that the primary forms of salbutamol in the plasma and urine samples from heifers are its sulfate and glucuronide conjugates.

Chiral liquid chromatography-mass spectrometry (LC-MS/MS) method development for the detection of salbutamol in urine samples.

A sequential solid-phase extraction (SPE) method was developed and validated using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the detection and quantification of salbutamol enantiomers in porcine urine. Porcine urine samples were hydrolysed with ß-glucuronidase/arylsulfatase from Helix pomatia and then subjected to a double solid-phase extraction (SPE) first using the Abs-Elut Nexus SPE and then followed by the Bond Elut Phenylboronic Acid (PBA) SPE. The salbutamol enantiomers were separated using the Astec CHIROBIOTIC™ T HPLC column (3.0mm×100mm; 5µm) maintained at 15°C with a 15min isocratic run at a flow rate of 0.4mL/min. The mobile phase constituted of 5mM ammonium formate in methanol. Salbutamol and salbutamol-tert-butyl-d9 (internal standard, IS) was monitored and quantified with the multiple reaction monitoring (MRM) mode. The method showed good linearity for the range of 0.1-10ng/mL with limit of quantification at 0.3ng/mL. Analysis of the QC samples showed intra- and inter-assay precisions to be less than 5.04%, and recovery ranging from 83.82 to 102.33%.

LC-MS-Based Metabolomics Discovers Purine Endogenous Associations with Low-Dose Salbutamol in Urine Collected for Antidoping Tests.

Current antidoping analytical methods are tailored mainly to the targeting of known drugs and endogenous molecules. This causes difficulties in rapidly reacting to emerging threats, such as designer drugs, biological therapeutic agents, and technologies. Biomarkers are considered as a promising approach for the fight against these threats to sport. The main purpose of this study was to find surrogate biomarkers induced by the intake of small amounts of the model compound salbutamol and explore a sensitive approach to help screen for possible drug misuse. Urine samples (91) from athletes with detectable salbutamol (30) and negative samples (61) were analyzed using a UHPLC-MS. A third group (30) was created by spiking salbutamol into negative samples to eliminate confounding effects. Data were then analyzed in XCMS to extract metabolic features. Orthogonal partial least squares-discriminant analysis was performed to select features correlated with detectable salbutamol (p(corr) > 0.5) and ROC analysis was performed to measure the predictive potential of the markers. Univariate analysis including Mann-Whitney U test and Spearman's correlation was conducted on selected markers. A total of 7000 metabolic features were parsed, one feature identified as hypoxanthine increased with salbutamol (p < 0.001). The ROC curve of hypoxanthine returned an AUC of 0.79 (p < 0.001). Correlation with salbutamol (r = 0.415, p < 0.01, Spearman's correlation) showed hypoxanthine and purine metabolism have association with salbutamol administration. This surrogate discovery approach needs further PK studies but in the meantime can be used as an intelligence-based complementary approach for targeting of athletes to be further tested.