Detection of capromorelin in urine following oral and dermal routes of administration.

Capromorelin is a growth hormone secretagogue. Despite promising results to alleviate muscle-wasting in the elderly, it has not advanced further in human development. Subsequent studies demonstrated capromorelin's ability to increase food intake in animals, leading to approval in the United States and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate growth hormone production and enhance performance. However, given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an adverse analytical finding (AAF) by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 µg of capromorelin orally (equivalent to 1 µL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 h after administration with a maximum urinary concentration of 7 ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg or 1 mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7 ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported.

δ13 C values of urinary 19-norandrosterone in antidoping samples and potential for adverse findings from boar offal consumption.

19-Norandrosterone (19NA) is the preferred urinary target compound to identify doping with nandrolone or related 19-norsteroids. At concentrations between 2.5 and 15 ng/mL, isotope ratio mass spectrometry (IRMS) is required to establish exogenous origin of urinary 19NA. An absolute difference of 3‰ between urinary 19NA and an endogenous reference compound (ERC) constitutes a finding for exogenous origin of 19NA. Over the last 3 years, 77 samples containing urinary 19NA between 2.5 and 15 ng/mL were analyzed at our laboratory. The measured δ13 C values for 19NA ranged from -29.5‰ to -16.8‰. In comparison, the δ13 C values for the corresponding urinary ERCs ranged from -22.4‰ to -16.2‰. Due to the considerable overlap in values between the target compound and the natural range of urinary ERCs, it can be challenging to distinguish between endogenous and exogenous origins of urinary 19NA. In addition, it is well known that consumption of offal from non-castrated pigs can produce 19NA in urine. To determine whether this could cause a positive IRMS finding under the current IRMS positivity criteria, meat from non-castrated boars fed a mixture of corn and soy was consumed by 13 volunteers. Two volunteers produced 19NA findings above 2.5 ng/mL, and the measured isotope values, while inconsistent with documented 19-norsteroid preparations, did meet IRMS positivity criteria. However, these increases in 19NA urinary concentrations were short-lived due to rapid elimination. Timely follow-up collections may help support a claim for dietary exposure when low urinary concentrations of 19NA with pseudo-endogenous isotope values are observed.

AICAr to SAICAr ratio can serve as additional marker of AICAr use.

AICAr (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, commonly referred to as AICAR) is an adenosine monophosphate-activated protein kinase agonist previously investigated for its therapeutic potential which has been shown to improve exercise performance in laboratory animals. For this reason, the World Anti-Doping Agency prohibits the use of AICAr in sports. AICAr can easily be detected by means of liquid chromatography-mass spectrometry, but being an endogenous metabolite, it cannot be discriminated from AICAr of a non-natural origin. Population-based concentration thresholds have been suggested as a means to identify suspicious samples that would require further analysis by carbon isotope ratio mass spectrometry (CIR); however, it remains at the discretion of the laboratory how to apply them. Here, the urinary ratio of AICAr to SAICA-riboside (SAICAr) that is a closely related purine metabolite was investigated. In an athlete population of 5517 samples, this ratio was relatively narrowly distributed with median values and 99th percentiles of 3.3 and 9.3, and 4.2 and 14 in male and female athletes, respectively. Analysis of urine samples obtained from an AICAr administration study demonstrated that the AICAr/SAICAr ratio can serve in addition to AICAr concentration as a valuable diagnostic trigger for follow-up analysis by CIR. Conceivably, this combination can offer better retrospectivity than AICAr concentration alone by allowing to decrease the AICAr concentration threshold without significantly increasing the number of suspicious samples.

Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10 µg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

Chromatographic-mass spectrometric analysis of the urinary metabolite profile of chlorphenesin observed after dermal application of chlorphenesin-containing sunscreen.

RATIONALE: Chlorphenesin is an approved biocide frequently used in cosmetics, and its carbamate ester is an approved skeletal muscle relaxant in certain countries for the treatment of discomfort related to skeletal muscle trauma and inflammation. A major urinary metabolite is 4-chlorophenoxy acetic acid (4-CPA), also known as para-chlorophenoxyacetate, which is also employed as a target analyte in sports drug testing to detect the use of the prohibited nootropic stimulant meclofenoxate. To distinguish between 4-CPA resulting from chlorphenesin, chlorphenesin carbamate, and meclofenoxate, urinary metabolite profiles of chlorphenesin after legitimate use were investigated. METHODS: Human administration studies with commercially available sunscreen containing 0.25% by weight of chlorphenesin were conducted. Six study participants dermally applied 8 g of sunscreen and collected urine samples before and up to 7 days after application. Another set of six study participants applied 8 g of sunscreen on three consecutive days, and urine samples were also taken for up to 5 days after the last dosing. Urine specimens were analyzed using liquid chromatography-high resolution (tandem) mass spectrometry, and urinary metabolites were identified in accordance with literature data by accurate mass analysis of respective precursor and characteristic product ions. RESULTS: In accordance with literature data, chlorphenesin yielded the characteristic urinary metabolites, chlorphenesin glucuronide, chlorphenesin sulfate, and 3-(4-chlorophenoxy)-2-hydroxypropanoic acid (4-CPP), as well as the common metabolite 4-CPA. 4-CPA and 4-CPP were observed at similar abundances, with urinary concentrations of 4-CPA reaching up to ~1500 and 2300 ng/mL after single and multiple sunscreen applications, respectively. CONCLUSION: 4-CPA is a common metabolite of meclofenoxate, chlorphenesin, and chlorphenesin carbamate. Monitoring the diagnostic urinary metabolites of chlorphenesin provides conclusive supporting evidence of whether chlorphenesin or the prohibited nootropic meclofenoxate was administered.

Measurement of urinary cobalt as its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol by liquid chromatography-tandem mass spectrometry for the purpose of anti-doping control.

Cobalt is well known for its ability to stimulate erythropoiesis via stabilization of hypoxia-inducible factors. In sports, this can provide a competitive benefit to athletes, so the World Anti-Doping Agency prohibits the use of cobalt in any form except its cobalamin vitamers. As of now, cobalt in biological fluids is detected by inductively coupled plasma mass spectrometry (ICP-MS), a technique which has very limited availability in anti-doping laboratories. Therefore, a quantitative method based on liquid chromatography-tandem mass spectrometry capable of measuring urinary cobalt in the form of its complex with 2-(5-chloro-2-pyridylazo)-5-diethylaminophenol (5-Cl-PADAP) has been developed and validated. A cobalt complex with deuterium-labeled 5-Cl-PADAP was used as internal standard. The method was found linear over the concentration range of 5-500 ng/ml with a combined standard uncertainty less than 10% at 15, 200, and 450 ng/ml. Stability of cobalt ions in urine was investigated over the course of 2 months; the concentration of free Co2+ was observed to decline by approximately 50% but restored upon hydrolysis with hydrochloric acid. Unlike ICP-MS, this method is practically unaffected by the presence of cyanocobalamin as the latter is resistant to acid hydrolysis. Notwithstanding the lack of formalized threshold concentration of cobalt in urine, it is highly desirable that more anti-doping laboratories engage in testing for cobalt levels to better understand the prevalence of cobalt misuse in athletes. Given that cobalt salts are inexpensive and easily obtainable, the risk of such abuse should not be underestimated.

High-throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction.

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high-throughput assay based on liquid chromatography-triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96-well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5-ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96-well collection plate. With a 10-min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti-Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02-0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

Inter-Laboratory Agreement of Insulin-like Growth Factor 1 Concentrations Measured Intact by Mass Spectrometry.

BACKGROUND: Insulin-like growth factor-I (IGF-1) is measured mainly by immunoassay for the diagnosis and treatment of growth hormone (GH) disorders, and to detect misuse of GH in sport. Immunoassays often have insufficient inter-laboratory agreement, especially between commercial kits. Over the expected range of IGF-1 in blood (∼50-500 ng/mL), in an inter-laboratory study we previously established a measurement imprecision of 11% (%CV) for the digested protein analyzed by LC-MS. Measuring intact IGF-1 by LC-MS should be simpler. However, no inter-laboratory agreement has been published. METHODS: Intact and trypsin-digested IGF-1 in 32 serum samples from healthy volunteers and human growth hormone administration studies were analyzed by LC-MS using different instruments in five laboratories, as well as by immunoassay in a single laboratory. Another 100 samples were analyzed for IGF-1, both intact and after trypsin-digestion, in each laboratory by LC-MS. The statistical relationship between measurements and the imprecision of each assay group was assessed. RESULTS: An intra-laboratory variability of 2-4% CV was obtained. Inter-laboratory variability was greater at 14.5% CV. Orthogonal regression of intact versus trypsin-digestion methods (n = 646) gave a slope of 1.01 and intercept of 2.05 ng/mL. CONCLUSIONS: LC-MS measurements of IGF-1 by intact and trypsin-digestion methods are not statistically different and each is similar to immunoassay. The two LC-MS approaches may be used interchangeably or together to eliminate concerns regarding an immunoassay IGF-1 measurement. Because intact and digested IGF-1 measurements generally agreed within 20% of each other, we propose this as a criterion of assay acceptability.

Urinary concentrations of AICAR and mannitol in athlete population.

Both AICAR and mannitol are prohibited for use in sports, but no decisive criteria that would guide anti-doping laboratories on data interpretation have been established so far. In an attempt to help harmonize reporting and management of analytical findings, reference population data collected for US athletes are presented. Upon analysis of 12 377 samples, mean urinary AICAR concentration was found to be 647 ± 365 ng/mL with median value of 574 ng/mL, 99th percentile at 1786 ng/mL and 99.7th percentile at 2151 ng/mL. Based on these results, we suggest that any sample with AICAR concentration greater than 2000 or 2500 ng/mL be analyzed by carbon isotope ratio mass spectrometry to establish the origin. Urinary mannitol concentrations demonstrate larger variation with the mean value of 72 ± 140 µg/mL and median at 41 µg/mL (n = 6407). While the 99.7th percentile for mannitol was measured to be 1094 µg/mL, the population data alone is not sufficient to suggest a threshold value. It is also shown that the use of mannitol as a sweetener in amounts of up to 20 g per day results in a urinary concentration of about 14 mg/mL. As only intravenous mannitol is prohibited in sports, controlled excretion studies are needed to see whether intravenous administration could in fact be discriminated from dietary intake. An important observation is that mannitol present in mg/mL quantities significantly increases urine specific gravity, which makes a widely accepted normalization approach not applicable.

Isotopically labeled boldenone as a better marker of derivatization efficiency for improved quality control in anti-doping analysis.

At present, anti-doping laboratories use androsterone, a major urinary steroid metabolite, to evaluate completeness of the derivatization step. This is typically done by calculating the ratio of mono-trimethylsilyl (TMS) androsterone to the total mono- and di-TMS androsterone. Certain samples may show an elevated percentage of mono-TMS androsterone indicating a failed derivatization step. In such cases, the laboratory would have to repeat the analysis or perform other remedial actions to ensure that completeness of derivatization is achieved. We have noticed that a poorly derivatized positive control sample spiked with various target analytes has a disproportionally low abundance of the di-TMS derivatives of boldenone and 18-nor-17ß-hydroxymethyl-17α-methylandrosta-1,4,13-trien-3-one (methandienone long-term metabolite). A follow-up investigation confirmed that 1,4-diene-3-one steroids are more likely to fail during the trimethylsilylation step. To better control derivatization efficiency, 13 C3 -boldenone (13C-BLD) was incorporated into our routine procedure as an additional internal standard. Analysis of a large number of urine samples has shown that derivatization of 13C-BLD could be grossly incomplete even in cases when mono-TMS androsterone is well below 1%. In other words, one or both of boldenone and the long-term metabolite of methandienone could remain undetected unless the laboratory has the means to recognize samples where derivatization of 1,4-diene-3-one steroids failed.

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 ß-subunit (hCGß) and ß-subunit core fragment (hCGßcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGß and hCGßcf were 0.04 IU/L, 0.47 pmol/L and 0.16 pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGß and hCGßcf 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.

The influence of small doses of ethanol on the urinary testosterone to epitestosterone ratio in men and women.

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti-Doping Agency (WADA) cut-off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut-off to detect samples with elevated T/E values from ingestion of low doses of ethanol.

Detection of Stimulants and Narcotics by Liquid Chromatography-Tandem Mass Spectrometry and Gas Chromatography-Mass Spectrometry for Sports Doping Control.

Sports drug testing laboratories are required to detect several classes of compounds that are prohibited at all times, which include anabolic agents, peptide hormones, growth factors, beta-2 agonists, hormones and metabolic modulators, and diuretics/masking agents. Other classes of compounds such as stimulants, narcotics, cannabinoids, and glucocorticoids are also prohibited, but only when an athlete is in competition. A single class of compounds can contain a large number of prohibited substances and all of the compounds should be detected by the testing procedure. Since there are almost 70 stimulants on the prohibited list it can be a challenge to develop a single screening method that will optimally detect all the compounds. We describe a combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) testing method for detection of all the stimulants and narcotics on the World Anti-Doping Agency prohibited list. Urine for LC-MS/MS testing does not require sample pretreatment and is a direct dilute and shoot method. Urine samples for the GC-MS method require a liquid-liquid extraction followed by derivatization with trifluoroacetic anhydride.

Carbon isotope ratio mass spectrometry for detection of endogenous steroid use: a testing strategy.

Isotope ratio mass spectrometry (IRMS) testing is performed to determine if an atypical steroid profile is due to administration of an endogenous steroid. Androsterone (Andro) and etiocholanolone (Etio), and/or the androstanediols (5α- and 5ß-androstane-3α,17ß-diol) are typically analyzed by IRMS to determine the (13) C/(12) C ratio. The ratios of these target compounds are compared to the (13) C/(12) C ratio of an endogenous reference compound (ERC) such as 5ß-pregnane-3α,20α-diol (Pdiol). Concentrations of Andro and Etio are high so (13) C/(12) C ratios can easily be measured in most urine samples. Despite the potentially improved sensitivity of the androstanediols for detecting the use of some testosterone formulations, additional processing steps are often required that increase labour costs and turnaround times. Since this can be problematic when performing large numbers of IRMS measurements, we established thresholds for Andro and Etio that can be used to determine the need for additional androstanediol testing. Using these criteria, 105 out of 2639 urine samples exceeded the Andro and/or Etio thresholds, with 52 of these samples being positive based on Andro and Etio IRMS testing alone. The remaining 53 urine samples had androstanediol IRMS testing performed and 3 samples were positive based on the androstanediol results. A similar strategy was used to establish a threshold for Pdiol to identify athletes with relatively (13) C-depleted values so that an alternative ERC can be used to confirm or establish a true endogenous reference value. Adoption of a similar strategy by other laboratories can significantly reduce IRMS sample processing and analysis times, thereby increasing testing capacity.

Detection of prohibited substances by liquid chromatography tandem mass spectrometry for sports doping control.

Drug testing for sports doping control programs is extensive and includes numerous classes of banned compounds including anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, diuretics, various peptide hormones, and growth factors. During competition, additional compounds may also be prohibited such as stimulants, narcotics, cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level of competition. Each of these classes of compounds can contain many prohibited substances that must be identified during the testing procedure. Various methods that have been designed to detect a large number of compounds in different drug classes are highly desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid methods have been described to simultaneously detect different classes of compounds. Here, we describe a simple urine sample cleanup procedure that can be used to detect numerous anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

The influence of diet on isotope ratio mass spectrometry values.

OBJECTIVES: Athletes have increasingly used testosterone (T) and other endogenous anabolic steroids that cannot be detected by conventional gas chromatography-mass spectrometry. This led to gas chromatography-combustion-isotope ratio mass spectrometry(GC/C/IRMS), which measures the relative amount of 13C in urinary steroids. Because exogenous testosterone is relatively low in 13C content, this study will determine if consuming a diet low in 13C plants, such as soy, can be confused with a GC/C/IRMS-positive test for exogenous testosterone. DESIGN: Cross-sectional study in which 22 vegetarians known to consume a diet depleted of 13C isotope were compared with a geographic control group of 14 subjects consuming a normal diet. SETTING: Two distinct subject populations with respect to diet. SUBJECTS: Subjects were recruited from a soy-based cooperative and control volunteers. Twenty-two of 24 research subjects completed the protocol compared with 14 of 22 control subjects. INTERVENTIONS: Independent variables were delta13C IRMS values,urinary steroid profile, and isoflavone analysis. MAIN OUTCOME MEASURES: Comparisons were made with respect to dietary analysis, isoflavones, and urinary steroid measurements using GC-C-IRMS. RESULTS: The delta13C values for 2 major metabolites of T (androsterone and etiocholanolone) were lower for the vegetarians than the controls (P = 0.005). The vegetarians excreted a median of 23 micromol/d of total isoflavones compared with 2.7 micromol/d for the control group (P =0.0002). CONCLUSIONS: The carbon isotope ratios of urinary testosterone metabolites of vegetarians consuming a diet that is markedly depleted of 13C content were lower than that of control subjects, but not low enough to result in World Anti-Doping Agency criteria for a positive IRMS analysis.

Another designer steroid: discovery, synthesis, and detection of 'madol' in urine.

Madol (17alpha-methyl-5alpha-androst-2-en-17beta-ol) was identified in an oily product received by our laboratory in the context of our investigations of designer steroids. The product allegedly contained an anabolic steroid not screened for in routine sport doping control urine tests. Madol was synthesized by Grignard methylation of 5alpha-androst-2-en-17-one and characterized by mass spectrometry and NMR spectroscopy. We developed a method for rapid screening of urine samples by gas chromatography/mass spectrometry (GC/MS) of trimethylsilylated madol (monitoring m/z 143, 270, and 345). A baboon administration study showed that madol and a metabolite are excreted in urine. In vitro incubation with human liver microsomes yielded the same metabolite. Madol is only the third steroid never commercially marketed to be found in the context of performance-enhancing drugs in sports.