Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction–tandem mass spectrometry to detect hCG use

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti‐Doping Agency (WADA) list of prohibited substances. The majority of WADA‐accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study we measured the concentration of intact hCG, free β‐subunit (hCGβ) and β‐subunit core fragment (hCGβcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC–MS/MS method. Mean concentrations of intact hCG, hCGβ and hCGβcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5^th percentile) for intact hCG, hCGβ and hCGβcf were 0.21 IU/L, 0.40 pmol/L, and 1.86 pmol/L, respectively. Based on these data, we recommend a threshold of 1.0 IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.     

Search for peptide immunogens of the β-subunit of human chorionic gonadotropin (hCG) capable of eliciting hormone specific and neutralizing antisera

The work reported herein describes our attempts to identify peptide immunogens of the β-subunit of the pregnancy-specific placental hormone, human chorionic gonadotropin (hCG), capable of eliciting hormone specific and neutralizing antisera. Hydrophilicity profiles of the β-subunits of hCG and the homologous pituitary hormone, human luteinizing hormone (hLH) were compared and two sequences which are hydrophilic but unique to hCG-β were identified. They are 6-12 and 109-119 of hCG-β. Results of the studies with the undecapeptide 1009-119 of hCG-β are reported in this paper. The undecapeptide amide was synthesized using the p-(acyloxy) benzhydrylamine resin and antisera to the peptide were elicited in rabbits using the peptide-diphtheria toxoid conjugate. The peptide is highly immunogenic as both the rabbits responded with high titers of antibodies to the peptide. The antipeptide antibodies bound to hCG but not to hLH showing thereby that the region 109-119 of hCG-β is a unique determinant of hCG. However, the antibodies were found not to neutralize the biological activity of hCG.     

人绒毛膜促性腺激素的临床应用进展

人绒毛膜促性腺激素（HCG）在临床上应用广泛,包括预测妊娠高血压综合征,提示异常妊娠的出现（如先兆流产、输卵管妊娠）,唐氏综合征的产前筛查,诊断和监测妊娠滋养层疾病（葡萄胎、绒癌等）,治疗先兆流产及习惯性流产,治疗小儿隐睾症及男性不育等。本文对公开发表的有关HCG临床应用的文献进行归纳、分析,总结了HCG近年来在妇产科疾病的预测、诊断及治疗方面的应用进展。     

The recombinant human chorionic gonadotropin prefilled pen: results of patient and nurse human factors usability testing

Objectives: The first prefilled pen for administration of recombinant human chorionic gonadotropin (r-hCG) has been developed. Usability testing was undertaken to evaluate the risk of dosing errors versus the existing r-hCG prefilled syringe, and assess function and handling of the pen.

Methods: Infertile women who were trying to conceive, and specialist nurses, were recruited in Germany. Usability goals were defined and categorized as critical or functional operational goals. Individual, non-interventional, standardized, usability tests (including ease-of-use assessment) were performed with patients and nurses. Cumulative test scores for critical operations were compared. Non-standardized qualitative analyses of nurse–patient training sessions were performed.

Results: The cumulative test score for the r-hCG prefilled pen was better than that of the existing prefilled syringe, so it was concluded that the overall risk of dosing errors was not higher with the pen. The ease of use of the pen was rated favorably by patients and nurses. Both user groups were confident that they could inject the correct dose using the pen.

Conclusions: The overall risk of dosing errors was not higher with the r-hCG prefilled pen than the existing prefilled syringe. The ease-of-use of the r-hCG prefilled pen was rated favorably by patients and nurses.     

PPARalpha agonists clofibrate and gemfibrozil inhibit cell growth, down-regulate hCG and up-regulate progesterone secretions in immortalized human trophoblast cells

We studied effects of PPARα agonists clofibric acid and gemfibrozil on cell growth and functions of immortalized human extravillous trophoblast cells. Levels of DNA and protein gradually increased during incubation for 4 days. Gemfibrozil (>0.25 mM) and clofibric acid (2.5 mM) suppressed the rate of increase in DNA and protein. Specific activities of fatty acyl-CoA oxidase and catalase were increased to about 1.2-2.0 times the control value by 0.05 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days. Acid phosphatase activity showed a small increase in response to both agents, but esterase activity changed little. The secretion of progesterone from the cells into the medium was increased by 0.25 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days, but that of human chorionic gonadotropin (hCG) was decreased by 0.35 mM gemfibrozil and 2.5 mM clofibric acid. The specific activity of lactate dehydrogenase in the cells was hardly changed at all after incubation for 3 days.These results suggest that gemfibrozil and clofibric acid inhibit the proliferation of trophoblast cells. Cell metabolism is probably affected by both agents. The two agents may down-regulate hCG and up-regulate progesterone secretions.     

先兆流产中绒毛膜促性腺激素与游离β亚单位及黄体酮的表达及意义分析

目的：探讨先兆流产患者绒毛膜促性腺激素（β-HCG）与游离β亚单位（fβ-HCG）及黄体酮含量（Progesterone, P）的表达及意义。方法先兆流产患者50例,设为观察组,正常早孕妇女50例为对照组,采用化学发光分析法对两组研究对象血样进行β-HCG、fβ-HCG、P含量的检测,每隔48 h对研究对象进行同样条件下的β-HCG、fβ-HCG、P含量的检测。48 h倍增率=（48 h测定值-初测值）＆#247;初测值。结果β-HCG、P含量组间比较差异无统计学意义（P〉0.05）, fβ-HCG含量观察组显著高于对照组,差异有统计学意义（P〈0.05）。β-HCG、fβ-HCG 48 h倍增率比较,差异无统计学意义（P〉0.05）。结论检测并综合分析β-HCG、fβ-HCG及黄体酮在先兆流产中的表达,对判断妊娠结局具有参考价值。     

Levels of urinary human chorionic gonadotrophin (hCG) following conception and variability of menstrual cycle length in a cohort of women attempting to conceive

ABSTRACT

Objectives: To define the variability of menstrual cycle length and contribution of follicular and luteal phases to overall cycle variability, and to examine the rise in urinary hCG in early pregnancy.

Methods: Menstrual cycle study. Urine samples from 101 women (recruited from two south-east counties in the UK) were assayed to determine day of luteinising hormone (LH) surge, lengths of follicular and luteal phases and correlations with total menstrual cycle length. HCG study. Daily urine samples collected from 86 women prior to conception until 43 days post-conception were assayed for hCG and examined versus time since LH surge, determined using fertility test kits.

Results: Mean menstrual cycle length was 27.7?±?3.4 days, mean follicular phase length was 14.5?±?3.4 days and mean luteal phase length was 13.2?±?1.9 days. Total cycle lengths varied between and within women. There was a significant correlation (r²?=?0.70) between follicular phase length and total cycle length; luteal phase length was less variable and showed no association with total cycle length. Concentrations of hCG were significantly similar between women when referenced against the day since LH surge. Three thresholds were determined to indicate time since conception as 1–2 weeks, 2–3 weeks and 3+ weeks.

Conclusions: Total cycle length variation is mainly determined by follicular phase variation and predicting menses onset to estimate time of pregnancy testing is unreliable. Evaluating concentrations of hCG relative to LH surge results in consistent increases between women up to 21 days after conception. Therefore, urinary hCG concentration can be used to accurately estimate time since conception.     

No effect of human chorionic gonadotropin treatment due to threatened abortion in early pregnancy for birth outcomes

Human chorionic gonadotropin (HCG) is used parenterally for treatment of threatened abortions and repeated spontaneous abortion in pregnant women. No controlled epidemiological studies of preterm birth and low birthweight newborns in pregnant women with HCG treatment have been published while the results of animal investigations were controversial. The data of 97 pregnant women with HCG treatment in the second and third months of pregnancy due to threatened abortion and/or previous spontaneous abortion(s) was compared with the data of other 38,054 pregnant women in the population-based large data set of the Hungarian Case-Control Surveillance of Congenital Abnormalities. There was no difference in mean gestational age at delivery and birth weight, in addition the rate of preterm birth and low birthweight newborns. Parenteral HCG treatment in the early pregnancy due to threatened abortion did not associate with a higher risk for preterm births or low birthweight newborns. However, a higher occurrence of gestational diabetes was found in pregnant women with HCG treatment and there was a slight male excess among newborn infants (p=0.06).     

人绒毛膜促性腺激素在冻融胚胎移植激素替代周期内膜转化日的应用

目的 探讨冻融胚胎移植(FET)激素替代周期(HRT)内膜转化日低黄体生成素(LH)时肌注人绒毛膜促性腺激素(hCG)对妊娠结局的影响.方法 回顾性分析2018年1月至2019年12月在皖北煤电集团总医院生殖医学科行FET激素替代方案助孕共271个周期的临床资料.根据内膜转化日低LH时是否肌注hCG分为hCG组(45周...     

Gonadotropins in doping: pharmacological basis and detection of illicit use

Parenteral administration of human chorionic gonadotropin (hCG) or luteinizing hormone (LH) stimulates the production of testosterone in males and these gonadotropins can therefore be used by athletes to enhance muscle strength. However, they are more expensive and less efficient than testosterone and anabolic steroids. Therefore their main use is probably to stimulate gonadal testosterone production during and after self-administration of testosterone or anabolic steroids. A positive effect of hCG on muscle strength has not been demonstrated in women and elevated concentrations of hCG in females are often caused by pregnancy. The use of gonadotropins is therefore prohibited only in males but not in females. HCG occurs at low but measurable concentrations in plasma and urine of healthy males and can be measured by sensitive methods. However, the characteristics of the method to be used for doping control have not been defined. Virtually all commercially available hCG assays have been designed for determination of hCG in serum rather than urine, which is used for doping control. Methods based on mass spectrometric detection of fragments derived from hCG extracted from urine by immunoadsorption have been developed but their suitability for doping control remains to be determined. The concentrations of LH in serum and urine are variable and more then 10-fold higher than those hCG. It is therefore difficult to detect illicit use of LH. The characteristics and reference values for hCG and LH assays used in doping control and the cutoff values need to be defined.     

Application of a non-target variable data independent workflow (vDIA) for the screening of prohibited substances in doping control testing

A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits of the workflow. Additional targets fulfilling the above requirements can be added without changing any settings. The performance of the vDIA workflow was illustrated by applying it to two screening methods in horse urine, with one workflow covering 331 basic drugs and the other covering 45 quaternary ammonium drugs (QADs). Both screening methods have good detection sensitivity with 84% of the basic drugs having Limits of Detection (LoDs) of ≤ 1 ng/mL and 84% of the QADs having LoDs of ≤ 0.4 ng/mL. Other method characteristics including retention reproducibility, method precision and false hit rate will also be presented.     

Screening and confirmatory analysis of recombinant human erythropoietin for racing camels' doping control

Recombinant human erythropoietin (rHuEPO) belongs to the therapeutic class of erythropoiesis stimulating agents (ESAs) due to its implication in the creation pathway of red blood cells and thus enhancement of oxygenation. Because of this bioactivity, rHuEPO has been considered as a major doping agent in sports competitions for decades. Over the years, doping control laboratories designed several analytical strategies applied to human and animal samples to highlight any misuse. Even though multiple analytical approaches have been reported, none has yet been dedicated to racing camels. Here, we describe an analytical strategy to test camel plasma samples at screening using an ELISA assay and a targeted nano‐liquid chromatography–high‐resolution tandem mass spectrometry for confirmatory analysis. The method was validated and has been successfully applied to post‐race samples, allowing the detection of a positive case of rHuEPO administration.     

New oxymesterone metabolites in human by gas chromatography‐tandem mass spectrometry and their application for doping control

Oxymesterone (17α‐methyl‐4, 17β‐dihydroxy‐androst‐4‐ene‐3‐one) is one of the anabolic androgenic steroids (AAS) banned by the World Anti‐Doping Agency (WADA). The biotransformation of oxymesterone is performed in vitro by human heptocytes and human urinary metabolic profiles are investigated after single dose of 20 mg to two adult males as well. Cell cultures and urine samples were hydrolyzed by β‐glucuronidase, extracted, and reacted with N‐Methyl‐N‐trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide (NH₄I), and dithioerythritol. After derivatization, a gas chromatography triple quadruple tandem mass spectrometry (GC‐MS/MS) using full scan and MS/MS modes was applied. The total ion chromatographs of the blank and the positive samples are compared, and 7 new metabolites were found. In addition to the well‐known 17‐epioxymesterone, oxymesterone is metabolized by 4‐ene‐reduction, 3‐keto‐reduction, 11β‐hydroxylation, and 16ξ‐hydroxylation. Based on the behavior of the MS/MS results of product ion and precursor ion modes, a GC‐MS/MS method has been developed monitoring these metabolites. The structures of metabolite 2 and 4 are tentatively identified as 17α‐methyl‐3β, 17β‐dihydroxy‐5α‐androstane‐4‐one and 17α‐methyl‐3α, 4ξ, 17β‐trihydroxy‐5α‐androstane, respectively. Detection of oxymesterone using new metabolites M2 and M4 can extend the detection window up to 4 days since the parent steroid was not detectable. Copyright © 2015 John Wiley & Sons, Ltd.     

A comparative evaluation of ELISA and latex agglutination tests for detection of human chorionic gonadotropin (hCG) in urine for pregnancy

To resolve questions about the relative sensitivity and specificity of the latex agglutination and enzyme-linked immunosorbent assay (ELISA) for the detection of human chorionic gonadotropin (hCG) in pregnancy, urine samples from 265 cases of suspected pregnancy were tested simultaneously by both methods. Included in the sample were women whose menstrual period ranged from 5-60 days late. In 243 cases (92%), identical results were obtained with both tests (142 positive and 101 negative). In an additional 12 cases, urine samples containing low levels of hCG when tested by the latex agglutination test showed negative results, while the same samples showed positive pregnancy (subsequently confirmed clinically) when tested by ELISA. Finally, 10 urine samples with red blood cells or pus cells gave false positive results on the latex agglutination test, but these cells did not interfere in the ELISA test. The lag period after the last menstrual period was 5-7 days for ELISA compared with 20 days for the latex agglutination test. The sensitivity of the former test was 0.5 IU hCG/ml of urine and 3.5 IU hCG/ml of urine for the latter test. Other advantages of the ELISA test are its stability (reagents are stable at room temperature, while those of the latex agglutination test are not), and its very clear and easy to read end point. The only advantage of the latex agglutination test was the time required for performing the test (5 minutes, compared with 40-50 minutes for ELISA).     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its β‐subunit: possible involvement of the disulfide bonds Cys9−Cys57 and Cys23−Cys72 in receptor binding of the hormone

Abstract: Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The α‐ and β‐subunits of hCG are highly cross‐linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGβ in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGβ were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [¹²⁵I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9?57) was found to be ≈ 4‐fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23?72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys⁹?Cys⁵⁷ and Cys²³?Cys⁷² of the β‐subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Administration study of recombinant human relaxin‐2 in horse for doping control purpose

The insulin‐like peptide relaxin (RLX), an endogenous peptide hormone produced in human for pregnancy and reproduction, is also known to exert a range of physiological and pathological effects. Its use is banned in human sports, horseracing, and equestrian competitions due to its potential performance enhancing effect through vasodilation resulting in the increase of blood and oxygen supplies to muscles. Little is known about the biotransformation and elimination of RLX in horses. This paper describes an administration study of rhRLX‐2 and its elimination in horses, and the development of sensitive methods for the detection and confirmation of rhRLX‐2 in both horse plasma and urine by nano‐liquid chromatography/high resolution mass spectrometry (nano‐LC/HRMS) after immunoaffinity extraction with the objective of controlling the abuse of rhRLX‐2 in horses. The limits of detection in plasma and urine are 2 pg/mL and 5 pg/mL, respectively. Two thoroughbred geldings were each administered one dose of 10 mg rhRLX‐2 subcutaneously daily for 3 consecutive days. The rhRLX‐2 could be detected and confirmed in the plasma and urine samples collected 105 h and 80 h, respectively, after the last dose of administration. For doping control purposes, rhRLX‐2 ELISA could be used as a screening test to identify potential positive samples for further investigation using the nano‐LC/HRMS methods.     

Studies on the in vivo metabolism of methylstenbolone and detection of novel long term metabolites for doping control analysis

The anabolic‐androgenic steroid methylstenbolone (MSTEN; 2α,17α‐dimethyl‐17β‐hydroxy‐5α‐androst‐1‐en‐3‐one) is available as a so‐called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of methylstenbolone. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained by the advertised metabolic stability of methylstenbolone. In 2013, a first investigation into the human metabolism of methylstenbolone was published, and two hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for methylstenbolone, and in the light of additional data originating from a recent publication on the in vivo metabolism of methylstenbolone in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated methylstenbolone together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled methylstenbolone, urine samples were collected for 29 days. Up to 40 different deuterated methylstenbolone metabolites were detected in post‐administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides methylstenbolone excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3β,17β‐diol and 2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3α,17β‐diol) were synthesized and characterized by NMR.     

Assay validation for vilanterol and its metabolites in human urine with application for doping control analysis

A bioanalytical method for detecting the ultra-long-acting beta₂-agonist (U-LABA) inhaled vilanterol and its metabolites, GSK932009 and GW630200, in urine was developed to potentially monitor permitted therapeutic versus prohibited supratherapeutic use in sport. The World Anti-Doping Agency (WADA) has established urinary concentration thresholds for the beta₂-agonists salbutamol and formoterol. Therapeutic use of vilanterol (25 μg once daily) was recently permitted by WADA; however, there is no established decision limit for adverse analytical findings due to insufficient urine concentration data. In this study, we validated an assay to detect vilanterol in urine collected from four healthy male and female athletes 0–72 h who received inhaled corticosteroid fluticasone furoate/U-LABA vilanterol (800/100 μg) combination, four times the normal therapeutic dose. After administration, subjects performed 1 h of bike ergometer exercise. The experiment was conducted again after repeat dosing for 1 week. Our method utilised liquid chromatography with tandem mass spectrometry and was validated over urine concentrations of 5–5000 (vilanterol) and 50–50,000 pg/ml (GSK932009 and GW630200). Plasma samples were analysed for vilanterol, using a previously validated assay. The peak concentration values for urine vilanterol, GSK932009 and GW630200 were 9.5, 10.4 and 0.17 ng/ml, for single dosing, and 18.6, 19.5 and 0.20 ng/ml, for repeat dosing. Urine samples from four volunteers using the final validated method are reported, demonstrating this assay has sensitivity to detect vilanterol or GSK932009 in urine for ≥72 h post single or repeat dosing with 800/100 μg fluticasone furoate/vilanterol, whereas GW630200 was quantifiable ≤4 h post dose.     

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses

The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.     

Quantitative detection of inhaled formoterol in human urine and relevance to doping control analysis

Formoterol is a frequently prescribed β₂‐agonist used for the treatment of asthma. Due to performance‐enhancing effects of some β₂‐agonists, formoterol appears on the prohibited list, published by the World Anti‐doping Agency (WADA). Its therapeutic use is allowed but restricted to inhalation. Since the data on urinary concentrations originating from therapeutic use is limited, no discrimination can be made between use and misuse when a routine sample is found to contain formoterol. Therefore the urinary excretion of six volunteers after inhalation of 18 µg of formoterol was investigated. A liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of formoterol in urine samples. Sample preparation consists of an enzymatic hydrolysis of the urine samples, followed by a liquid‐liquid extraction at pH 9.5 with diethyl ether/isopropanol (5/1, v/v). Analysis was performed using selected reaction monitoring after electrospray ionization. The method was linear in the range of 0.5–50 ng/ml. The limit of quantification (LOQ) was 0.5 ng/ml. The bias ranged between ‐1.0 and ‐6.8 %. Results for the urinary excretion show that formoterol could be detected for 72 h. The maximum urinary concentration detected was 8.5 ng/ml without and 11.4 ng/ml after enzymatic hydrolysis. Cumulative data showed that maximum 11.5% and 23% of the administered dose is excreted as parent drug within the first 12 h, respectively, non‐conjugated and conjugated. Analysis of 82 routine doping samples, declared positive for formoterol during routine analysis, did not exhibit concentrations which could be attributed to misuse. Copyright © 2012 John Wiley & Sons, Ltd.