β‐2 Agonists as Misusing Drugs? Assessment of both Clenbuterol‐ and Salbutamol‐related European Medicines Agency Pharmacovigilance Database Reports

A recent years’ increase in misusing levels of image‐ and performance‐ enhancing drugs (IPEDs) has been observed. Out of these drugs, beta‐2 agonists have recently emerged for their potential of misuse, especially for slimming and bodybuilding purposes. To this perspective, clenbuterol (‘the size zero pill’) has been reported as being both popular and widely available from the illegal market. All clenbuterol and salbutamol misuse/abuse/dependence/withdrawal/overdose/off‐label spontaneous reports (2006–2016) from the European Medicines Agency (EMA) EudraVigilance (EV) database were collected and analysed by age range, gender, concomitant therapies and source of information. From the EV database, 55 of a total number of 920 ‘suspect’ misuse/abuse/dependence/withdrawal/overdose/off‐label ADRs (e.g. 5.97%; corresponding to 25 of 138 individuals) and 1310 of 62,879 ADRs (e.g. 2.08%; corresponding to 474 of 6923 individuals) were, respectively, associated with clenbuterol (typically ingested in combination with a range of anabolic steroids) and salbutamol. Proportional reporting ratio (PRR) value for misuse/abuse ADRs was higher (PRR = 18.38) for clenbuterol in comparison with salbutamol. Clenbuterol misuse/abuse could be a cause for major concern, especially in vulnerable individuals.     

Testing for AB‐PINACA in human hair: Distribution in head hair versus pubic hair

Synthetic cannabinoid receptor agonists were first identified in herbal products in 2008 advertised as a legal replacement for cannabis. These herbal incense are usually called “spice” and among these, one product in particular has gained popularity: AB‐PINACA (N‐[(2S)‐1‐Amino‐3‐methyl‐1‐oxobutan‐2‐yl]‐1‐pentyl‐1H‐indazole‐3‐carboxamide). This drug has been discovered to have a stronger binding to human cannabinoid CB₁ and CB₂ receptors than ?⁹‐THC.While some articles have been published regarding the presence of AB‐PINACA in biological fluids such as blood and urine, none reports the presence of AB‐PINACA in hair. We have developed and validated a method for detection of AB‐PINACA in hair using a liquid chromatography?tandem mass spectrometry system and applied it to head and pubic hair obtained in a case of intoxication. The validation procedure demonstrated a limit of detection and a limit of quantification of 0.5 and 1 pg/mg, respectively and acceptable linearity, repeatability, and reproducibility. AB‐PINACA tested positive in the blood (5.7 ng/mL) and less than 1 ng/mL was found in urine. The analysis of the hair specimens resulted in an unusual distribution of the drug between head and pubic hair. AB‐PINACA was identified at a higher concentration in head hair (195 pg/mg) versus in pubic hair (5 pg/mg). The very low concentration of AB‐PINACA in the urine after consumption, due to rapid metabolism, could explain this infrequent distribution, as pubic hair can be contaminated by urine. In any case, it cannot be excluded that the high concentration in head hair may be due to environmental contamination.     

Accumulation of β-agonists clenbuterol and salbutamol in black and white mouse hair

Hair has been shown to be an excellent site for the accumulation of different drugs including β-agonists, and therefore, it would be an appropriate matrix for surveillance for the presence of drug residues. The aim of this study was to determine concentrations and to compare accumulation of two different β-agonists in black and white mice hair by use of ELISA as a screening quantitative method. The study included 200 8-week-old white and black mice. One group of black mice and one group of white mice were treated with clenbuterol in a dose of 2.5 mg/kg body mass per os for 28 days. Other animals were treated in the same way with salbutamol. The highest (±SD) clenbuterol concentration of 631.4 ± 23.5 ng/g in black hair and 228.5 ± 156.2 ng/g in white hair was determined on day 1 of treatment withdrawal. Study results revealed the black-to-white hair ratio of clenbuterol accumulation to be 1:2-1:4 and of salbutamol accumulation 1:1.4. The mean (±SD) salbutamol concentrations determined on day 1 of treatment withdrawal was 23.9 ± 0.9 ng/g and 16.4 ± 1.1 ng/g in black and white hair samples, respectively. The study demonstrated that residues could be determined in hair samples even after a 30-day withdrawal period.     

Accumulation of the β<Subscript>2</Subscript>-adrenergic agonist clenbuterol in mouse dark hair

The aim of the present study was to evaluate the suitability of dark hair as a matrix for determination of the β₂-adrenergic agonist clenbuterol residues using previously validated enzyme-linked immunosorbent assay (ELISA) as a screening method for its quantitative determination. The experimental group of mice (n = 60) were treated with two different anabolic dosages of clenbuterol for 15 days, whereas the control group of animals (n = 30) was left completely untreated. Hair samples were collected on days 0, 5, 10, and 15 of treatment. Validation of the ELISA analytical procedure showed good recovery (mean recovery 74%) with an acceptable intra-assay variation in individual measurements for all hair samples to which 5, 10, and 50 ng/g clenbuterol were added (CV < 10%). Low blank levels of clenbuterol (2.4 ± 0.6 ng/g) were measured in hair of untreated mice, whereas significantly higher clenbuterol concentrations rising proportionally with the time of treatment were recorded in hair of mice treated with lower (6.5 mg/kg body mass) and higher (12.5 mg/kg body mass) dose of clenbuterol. The peak hair concentration of clenbuterol measured on the last day of treatment (day 15) was 1553.9 ± 140.1 ng/g and 6248.3 ± 589.4 ng/g in the lower and higher dose group, respectively. Study results clearly indicated dark hair as a pigmented tissue to have a high accumulation potential for clenbuterol residues, thus being the target matrix of choice for detection of clenbuterol abuse as an anabolic in meat production.     

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti‐Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO_2max and a 20‐km time‐trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0–24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol C_max was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·?h) than EX (16.9±2.9 ng/mL·?h)(P<0.01) and EXD (16.1±3.2 ng/mL·?h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of ractopamine in pig hair using liquid chromatography with tandem mass spectrometric detection

A quantitative analytical procedure for the determination of ractopamine in pig hair has been developed and validated. The hair samples were washed and incubated at 75 °C with isoxuprine and hair extraction buffer. The drug present was quantified using mixed solid-phase extraction and liquid chromatography with tandem mass spectrometric detection. The limit of quantization (LOQ) was 10 pg/mg and the intra-day precision at 25 pg/mg and 750 pg/mg was 0.49% and 2.8% respectively. Inter-day precision was 0.88% and 3.52% at the same concentrations. The hair extraction percentage recovery at 25 pg/mg and 50 ng/mL was 99.47% and 103.83% respectively. The extraction percentage recovery at 25 pg/mg and 50 ng/mg was 93.52% and 100.26% respectively. Our results showed that ractopamine residues persist in hair in 24 days of withdrawal and also showed the possibility to test ractopamine from pig hair samples.     

Separation of positional isomers of nine 2‐phenethylamine‐derived designer drugs by liquid chromatography–tandem mass spectrometry

The synthesis of positional isomers of designer drugs is a common way of bypassing legal restrictions. For forensic case work, and especially for the legal assessment of cases, there is a need for screening methods capable of the unequivocal identification of positional isomers. The presented liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) method facilitates separation of positional isomers of 9 2‐phenethylamine‐derived designer drugs in different matrices including seized materials, hair, serum, and urine specimens. Chromatographic separation was achieved on a biphenyl phase using gradient elution with a total runtime of 26 minutes. The limit of detection was 25 pg/mg for hair samples and ranged from 0.1 ng/mL to 0.5 ng/mL for serum and from 0.2 ng/mL to 1.2 ng/mL for urine samples. The method proved to be selective and sensitive and showed good chromatographic resolution (R ≥ 1.2). The method was successfully applied to routine case samples.     

Antagonism of Reserpine-induced Hypothermia in Mice by Some β-Adrenoceptor Agonists

Abstract: The reversal of reserpine-induced hypothermia in mice by three β-adrenoceptor agonists, terbutaline, salbutamol and clenbuterol, was examined. Terbutaline, 5 and 20 mg/kg intraperitoneally, caused a slight reversal of the hypothermia. Salbutamol, 5 mg/kg intraperitoneally, produced a pronounced and 1 mg/kg a slight reversal. Clenbuterol was very potent in reversing the hypothermic effect of reserpine with significant effect at 0.02 mg/kg intraperitoneally. Propranolol, 2.5 mg/kg intraperitoneally, and the selective β₂-receptor antagonist IPS 339, 5 mg/kg intraperitoneally, almost completely antagonized the effect of clenbuterol, 1 mg/kg intraperitoneally. The β-receptor antagonist metoprolol at 5 and 25 mg/kg intraperitoneally only partially antagonized the effect of clenbuterol. Since clenbuterol is much more lipophilic than salbutamol and terbutaline, it is suggested that the reserpine reversal produced by clenbuterol is at least partially of central origin and possibly mediated by stimulation of noradrenergic β₂ receptors.     

Proliferation of the human urothelium is induced by atypical β1‐adrenoceptors

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Evaluation of R‐ (−) and S‐ (+) Clenbuterol enantiomers during a doping cycle or continuous ingestion of contaminated meat using chiral liquid chromatography by LC‐TQ‐MS

Clenbuterol is known to improve competition resistance and muscular growth in athletes. Although it is an illegal drug, its use by farmers is widely spread to induce growth of their cattle. Thus, when clenbuterol is found in the urine of an athlete, there is doubt whether it was consumed with doping purposes or if it is due to the consumption of meat from a clenbuterol‐fed animal. Previous studies suggest that enantiomeric relationship of clenbuterol may be different according to the intake source. However, the enantiomeric relationship throughout a doping cycle or a continuous intake of contaminated meat has not yet been explored. In this first approximation, our aim was the development and validation of a sensitive and rapid method for the determination of S‐ (+) and R‐ (─) clenbuterol enantiomers to be used in a controlled study in rats fed for one week with contaminated meat or simulating a doping cycle. Enantiomers were measured using liquid chromatography coupled to mass spectrometry with a triple quadrupole analyzer (LC‐TQ‐MS) and were separated on an AGP Chiralpak column. The method was fully validated following the VICH (Veterinary International Conference on Harmonization guidelines) and was linear in the range of 12.5–800 pg/mL with a correlation coefficient of ≥0.98 for each enantiomer, and with a limit of quantitation and detection (LOQ and LOD) of 12.5 pg/mL and 6.5 pg/mL, respectively, for both enantiomers. The application of this method pointed out the shift of the enantiomeric relationship in urine from rats during the first five days of the doping cycle compared to those fed with contaminated meat. This finding can be of substantial importance in further doping studies.     

Single‐dose administration of clenbuterol is detectable in dried blood spots

Clenbuterol is a β₂‐agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time‐consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 μg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post‐ingestion, with additional urine collections on days 7 and 10. Using LC–MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post‐ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7–10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 μg.     

Method validation and preliminary pharmacokinetic studies on the new designer stimulant 3‐fluorophenmetrazine (3‐FPM)

Pharmaceutical research not only provides the basis for the development of new medicinal products but also for the synthesis of new drugs of abuse. 3‐Fluorophenmetrazine (3‐FPM), a fluorinated derivative of the anorectic phenmetrazine, was first patented in 2011 and appeared on the drug market in 2014. Though invented for potential medical purposes, pharmacokinetic data on this compound, crucial for interpreting forensic as well as clinical cases, are not available. Therefore, a liquid chromatography?electrospray ionization?tandem mass spectrometry (LC?ESI?MS/MS) method for the detection of 3‐FPM in serum, urine, and oral fluid was developed, validated for urine and serum, and used to quantify 3‐FPM in samples obtained during a controlled self‐experiment. The method proved to be linear, selective and sufficiently sensitive. The limits of detection (LODs) were 0.1 ng/mL, 0.2 ng/mL, and 0.05 ng/mL in serum, urine, and oral fluid. Inter‐day precision and intra‐day precision (RSD) in serum samples were below 6.3% and below 8.5%, respectively. The highest serum concentration (c_max) of 210 ng/mL was reached 2.5 hours (t_max) after ingestion. The elimination half‐life and the volume of distribution were calculated to be approx. 8.8 hours and 400 L (5.3 L/kg). 3‐FPM could be detected in serum and urine up to 82 hours and 116 hours, respectively. It was still detected in the last oral fluid sample taken 55 hours after ingestion. 3‐FPM was mainly excreted unchanged. Main metabolic reactions were aryl‐hydroxylation and N‐hydroxylation. Interestingly, the product of oxidative ring opening (2‐amino‐1‐(3‐fluorophenyl)propan‐1‐ol) showed the largest window of detection in the self‐experiment.     

Functional identification of β3‐adrenoceptors in the guinea‐pig ileum using the non‐selective β‐adrenoceptor antagonist (±)‐bupranolol

1 To clarify whether there is a species difference or a tissue difference in β₃‐adrenoceptors, the β₃‐adrenoceptors mediating relaxations to catecholamines ((–)‐isoprenaline, (–)‐noradrenaline and (–)‐adrenaline), a selective β₃‐adrenoceptor agonist BRL37344 and a non‐conventional partial β₃‐adrenoceptor agonist (±)‐CGP12177A (a potent β₁‐ and β₂‐adrenoceptor antagonist with a partial β₃‐adrenoceptor agonist property) were investigated in the guinea‐pig ileum. 2 Catecholamines and β₃‐adrenoceptor agonists induced concentration‐dependent relaxations of pre‐contracted strips of the guinea‐pig ileum. The rank order for their relaxing potency was (–)‐isoprenaline (pD₂: 7.60) > BRL37344 (7.05) > (–)‐noradrenaline (6.38) > (±)‐CGP12177A (6.25) > (–)‐adrenaline (6.07). 3 In the presence of the non‐selective β₁‐ and β₂‐adrenoceptor antagonist (±)‐propranolol (1 μM ), only small rightward shifts of the concentration–response curves (CRCs) to these agonists were observed and the rank order of potency of agonists was BRL37344 (pD₂: 7.00) > (±)‐CGP12177A (6.17) > (–)‐isoprenaline (6.01) > (–)‐noradrenaline (5.69) > (–)‐adrenaline (5.41). 4 In the presence of (±)‐propranolol (1 μM ), the additional presence of (±)‐bupranolol (3–30 μM ), a non‐selective β₁‐, β₂‐ and β₃‐adrenoceptor antagonist, caused a concentration‐dependent rightward shift of the CRCs to catecholamines and β₃‐adrenoceptor agonists. Schild plot analyses of (±)‐bupranolol against these agonists gave pA₂ values of 6.02 ((–)‐isoprenaline), 6.03 ((–)‐noradrenaline), 6.01 ((–)‐adrenaline), 6.56 (BRL37344) and 5.74 ((±)‐CGP12177A), respectively. All Schild plot slopes were not significantly different from unity. The pA₂ values of (±)‐bupranolol obtained for the guinea‐pig β₃‐adrenoceptors were about one log unit less than the values obtained for the rat β₃‐adrenoceptors and about two log units less than the values obtained for dog β₃‐adrenoceptors. 5 These results confirm that functional β₃‐adrenoceptors are present in the guinea‐pig ileum and that the relaxations of these agonists are mainly mediated via β₃‐adrenoceptors in this tissue. The differential antagonistic potency of (±)‐bupranolol may suggest that there is a species difference between the three species (guinea‐pig, dog and rat) in their β₃‐adrenoceptors.     

PTCA (1H‐pyrrole‐2,3,5‐tricarboxylic acid) as a marker for oxidative hair treatment

Hair analysis for the assessment of alcohol or drug abstinence has become a routine procedure in forensic toxicology. Hair coloration leading to loss of incorporated xenobiotics and to false negative results has turned out to be a major problem. Currently only colored extracts provide hints of manipulations but not bleaching. A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated to determine 1H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA), a major oxidation product of melanin. PTCA was determined in natural hair samples (n = 21) after treatment with 3% hydrogen peroxide (H₂O₂) for 30 or 40 minutes with concentrations up to 12% for 40 minutes. In another series, 12 natural hair samples were submitted to different coloration procedures (henna, tinting, semi‐permanent and permanent dyeing, bleaching) and the changes in PTCA content were determined. A significant increase in the PTCA content was found for both incubation times and increasing H₂O₂ concentrations. Coloration with henna or tinting had no influence on PTCA levels detected, but a significant increase was observed after semi‐permanent and permanent dyeing and bleaching. As PTCA concentrations in natural hair were found to be in a range of <2.1–16.4 ng/mg (8.4 ± 3.8 ng/mg, mean ± SD, n = 33), a cut‐off of 20 ng/mg is recommended for the distinction between natural vs. excessively oxidized hair. In case of naturally low melanin content (light‐blond or white hair), no marked increase in PTCA may occur. The present study demonstrated that PTCA is formed during oxidative treatment of melanin in hair, which can be used to detect previous hair coloration including oxidation.     

Enantioselective disposition of (R,R)‐formoterol, (S,S)‐formoterol and their respective glucuronides in urine following single inhaled dosing and application to doping control

Formoterol is a long‐acting beta2‐adrenoceptor agonist (LABA) used for the treatment of asthma and exercise‐induced bronchoconstriction. Formoterol is usually administered as a racemic (rac‐) mixture of (R,R)‐ and (S,S)‐enantiomers. While formoterol is restricted by the World Anti‐Doping Agency (WADA), inhalation of formoterol is permitted to a predetermined dose (54 μg/24 hours) and a urine threshold of 40 ng/mL. However, chiral switch enantiopure (R,R)‐formoterol is available, effectively doubling the therapeutic advantage for the same threshold. The aim of this study was to investigate whether formoterol exhibits enantioselective urinary pharmacokinetics following inhalation. Six healthy volunteers were administered a 12 μg inhaled dose of rac‐formoterol. Urine was collected over 24 hours and analyzed by enantioselective ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) assay. Total (free drug plus conjugated metabolite) median (min‐max) rac‐formoterol urine levels following inhalation were 1.96 (1.05–13.4) ng/mL, 1.67 (0.16–9.67) ng/mL, 0.45 (0.16–1.51) ng/mL, 0.61 (0.33–0.78) ng/mL, and 0.17 (0.08–1.06) ng/mL at 2, 4, 8, 12, and 24 hours, respectively, well below the 2019 urine threshold. The proportion of conjugation differed between enantiomers with glucuronide conjugation much greater for (R,R)‐formoterol (around 30%–60% of total) compared to (S,S)‐formoterol (0%–30%). There was clear evidence of inter‐individual enantioselectivity observed in the ratios of (R,R):(S,S)‐formoterol, where (S,S)‐ was predominant in free formoterol, and (R,R)‐ predominant in the conjugated metabolite. In conclusion, rac‐formoterol delivered by inhalation exhibits enantioselective elimination in urine following single‐dose administration. Enantioselective assays should be employed in doping control to screen for banned beta2‐agonist chiral switch products such as (R,R)‐formoterol, and total hydrolyzed rac‐formoterol is warranted to account for inter‐individual differences in enantioselective glucuronidation.     

Analysis of the beta 1 and beta 2 adrenoceptor interactions of the partial agonist, clenbuterol (NAB365), in the rat jugular vein and atria

Summary The potent bronchodilator, clenbuterol, was compared to other beta adrenoceptor agonists with regard to affinity and efficacy for interaction with beta₁ and beta₂ adrenoceptors in the rat jugular vein and atria. Clenbuterol was a potent partial beta adrenoceptor agonist in both tissues based on the following observations: 1. Maximal relaxation of the jugular vein and increases in atrial rate to clenbuterol were less than maximal responses to other beta adrenoceptor agonists. 2. Clenbuterol antagonized responses to the stronger agonist, isoproterenol, in both tissues and 3. the equilibrium dissociation constant for clenbuterol approximated the ED₅₀ concentration for vascular relaxation and increase in atrial rate, a characteristic of some, but not all, partial agonists. Relative to other beta adrenoceptor agonists, clenbuterol showed high affinity toward both beta₁ and beta₂ adrenoceptors and selectivity toward beta₂ adrenoceptors. Equilibrium dissociation constants were 38 and 6.3 nM for beta₁ and beta₂ adrenoceptors, respectively. The high affinity of clenbuterol toward beta₁ and beta₂ adrenoceptors was coupled to a low relative efficacy of clenbuterol to activate either beta₁ or beta₂ adrenoceptors. Most beta₂ adrenoceptor agonists such as isoproterenol or salbutamol require approximately 1–3% adrenoceptor occupation for 40–50% relaxation of the jugular vein whereas clenbuterol required approximately 100% adrenoceptor occupation for a similar response. Thus, based on our analysis, the high agonist potency of clenbuterol results primarily from the high affinity toward beta adrenoceptors rather than efficient activation of the adrenoceptor as occurs with isoproterenol or salbutamol.     

Additional role of serum 25‐hydroxyvitamin D3 levels in atherosclerosis in Chinese middle‐aged and elderly men

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Urine concentrations of oral salbutamol in samples collected after intense exercise in endurance athletes

Our objective was to investigate urine concentrations of 8 mg oral salbutamol in samples collected after intense exercise in endurance athletes. Nine male endurance athletes with a VO_2max of 70.2 ± 5.9 mL/min/kg (mean ± SD) took part in the study. Two hours after administration of 8 mg oral salbutamol, subjects performed submaximal exercise for 15 min followed by two, 2‐min exercise bouts at an intensity corresponding to 110% of VO_2max and a bout to exhaustion at same intensity. Urine samples were collected 4, 8, and 12 h following administration of salbutamol. Samples were analyzed by the Norwegian World Anti‐doping Agency (WADA) laboratory. Adjustment of urine concentrations of salbutamol to a urine specific gravity (USG) of 1.020 g/mL was compared with no adjustment according to WADA's technical documents. We observed greater (P = 0.01) urine concentrations of salbutamol 4 h after administration when samples were adjusted to a USG of 1.020 g/mL compared with no adjustment (3089 ± 911 vs. 1918 ± 1081 ng/mL). With the current urine decision limit of 1200 ng/mL for salbutamol on WADA's 2013 list of prohibited substances, fewer false negative urine samples were observed when adjusted to a USG of 1.020 g/mL compared with no adjustment. In conclusion, adjustment of urine samples to a USG of 1.020 g/mL decreases risk of false negative doping tests after administration of oral salbutamol. Adjusting urine samples for USG might be useful when evaluating urine concentrations of salbutamol in doping cases. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of ractopamine and salbutamol in pig hair by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometric method was developed for the determination of two β-agonists (ractopamine and salbutamol) in pig hair samples. An isotope of ractopamine-d5 or salbutamol-d6 as an internal standard was used to carry out quantitative analysis. Concentrated sodium hydroxide was used to pretreat hair samples and then purified by the solid phase extraction (SPE) procedure. The extracted solution was evaporated and reconstituted for injection in the instrument with electrospray ionization (ESI) operating in a positive multiple-reaction-monitoring (MRM) mode. Ractopamine and salbutamol separation were performed on C18 analytical column under gradient condition. The internal standard calibration curve was linear in the range of concentration from 0.5 to 100 ng mL⁻¹ (R² > 0.995). Recoveries of this method estimated at three spiked concentrations of 100, 250 and 500 ng mL⁻¹ in pig hair samples, were 79–82% for ractopamine and 77–96% for salbutamol. The corresponding inter-day and intra-day precisions expressed as relative standard deviation (RSD %) were 3.8–6.4% and 3.8–8.6%, respectively. The analytical time for one sample was 8 min. The detection limit of this method was 0.6 and 8.3 ng mL⁻¹ for ractopamine and salbutamol, respectively. This developed method can be applied for monitoring the use of the β-agonists salbutamol and ractopamine in swine feed incurred pig hair.     

A pharmacological study of bronchodilator properties of NKH477, forskolin,and β‐agonists on guinea pig and ovine isolated bronchioles

In this study we assessed the relaxant responses of two direct adenylate cyclase activators, NKH477 and forskolin, in comparison with two β‐adrenoceptor agonists, salbutamol and isoprenaline. The possible potentiation effect of NKH477 and forskolin on β‐agonist‐induced bronchodilatation was examined. The effectiveness of NKH477 and forskolin in reversing tachyphylaxis development to salbutamol or isoprenaline was also investigated. We tested the in vitro bronchodilator effect of salbutamol, NKH477, and forskolin (10^–9–10^–4 M) on isolated guinea pig bronchiolar ring segments precontracted with carbachol (3 μM). Salbutamol, NKH477, and forskolin produced a concentration‐dependent relaxation. Potency values (pD₂) were determined from cumulative concentration–response curves. The rank order for their potencies was salbutamol > NKH477 > forskolin (7.3 ± 0.3, 6.4 ± 0.3, and 5.4 ± 0.1, respectively). The bronchodilator effects of salbutamol, isoprenaline, NKH477, and forskolin (10^–9–10^–4 M) were examined on isolated ovine bronchioles precontracted with carbachol (0.3 μM). Isoprenaline, NKH477, and forskolin produced a concentration‐dependent relaxation with pD₂ values of 6.1 ± 0.2, 5.4 ± 0.2, and 5.3 ± 0.2, respectively. Tachyphylaxis to the relaxant effects of salbutamol on guinea pig isolated bronchioles was experimentally induced and the potency of salbutamol was reduced to 5.9 ± 0.2 after 24 h incubation with salbutamol (10^–5M). NKH477 and forskolin (10^–6 M) produced a partial reversal of tachyphylaxis to salbutamol‐induced relaxation using salbutamol pretreated tissues. The potency of salbutamol was increased to 6.6 ± 0.2 and 5.9 ± 0.2 after incubation with NKH477 or forskolin (10^–6 M), respectively. Tachyphylaxis to the relaxant effects of isoprenaline resulted in a reduced potency of 5.7 ± 0.2. Forskolin (10^–6 M) produced a partial reversal of tachyphylaxis, while NKH477 (10^–6 M) produced a complete reversal of tachyphylaxis to isoprenaline‐induced relaxation with an pD₂ value of 6.3 ± 0.1. In conclusion, the guinea pig and sheep isolated bronchioles serve as good models to study the relaxant effects of the bronchodilator agents salbutamol, isoprenaline, NKH477, and forskolin. The β‐agonists examined had higher potencies than NKH477 or forskolin. However, the two adenylate cyclase activators, with greater effectiveness of NKH477, when used in combination with the β‐agonists, could produce an increase in the potency of the β‐agonists. Furthermore, the effectiveness of NKH477 and forskolin in reversing tachyphylaxis to the bronchodilator effects of the β‐agonists, particularly salbutamol, may provide an advantage in long‐term use of β‐agonists in bronchial asthma therapy. Drug Dev. Res. 51:169–176, 2000. © 2001 Wiley‐Liss, Inc.