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Metal oxide-filled reactors constructed with ceramic tubes or fused silica capillary are widely used for combustion in gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS). However, they tend to be easily cracked or broken and prone to leaks at operating temperatures of ∼950 °C. Here we introduce a modified commercially available catalytic combustion/reduction methanizer to quantitatively convert organics to CO2 for δ13C analysis while retaining chromatographic resolution. These modified “ARC” reactors operate with a transition-metal catalyst that requires a flowing O2 gas to enable complete conversion to CO2 at lower temperature (620 °C) with acceptable reactor life, reduced complexity, and improved robustness. Performance of two versions of the ARC reactors with different combustion volumes was characterized by analysis of steroid and alkane isotopic standard materials. Linearity of steroid isotopic standards ranged from 0.02 to 0.60 ‰/V in the range of 25 to 200 ng of each steroid i...

The Goldman dilemma presented athletes with a Faustian bargain that guaranteed winning an Olympic gold medal in their sport but resulted in certain death 5 years later. Athletes’ responses to Goldman’s bargain were reported from 1982 to 1995. Several studies subsequently evaluated people’s willingness to accept the bargain proposed in the Goldman question. Our study updates Goldman’s question using contingent-behavior questions, a preference-elicitation method widely applied in economics, marketing and psychology to understand people’s choice behavior. Contingent-behavior questions ask people to evaluate hypothetical tradeoffs between outcomes when real-world decisions are unobservable, nonexistent, or unreliable. A web-enabled survey was conducted with athletes in 50 sports between June, 2012 and April, 2013. Athletes were invited by their sport governing bodies in the United States to complete the online survey. Responses from 2888 athletes were collected. Our reformulation elicited athletes’ willingness to accept a performance-enhancing drug (PED) associated with the risk of a realistic fatal event, not certain death. A double-bounded dichotomous-choice question format was used to elicit athletes’ maximum acceptable mortality risk (MAMR) for winning an Olympic gold medal. Data were analyzed using an interval regression model to estimate the implicit probability of accepting a continuous risk level. MAMR was defined as the mortality risk level with a 0.50 probability of acceptance. Estimated mean MAMRs varied between 7 and 14% across athletes in different ranks and sports. Elite athletes were generally the most willing to accept a fatal cardiovascular risk to win a gold medal in the Olympics. This range was similar to the levels of risk that patients accept for life-changing interventions. Results suggest that very few athletes would be expected to accept a PED in the bargain postulated by the Goldman dilemma. Risk tolerance among elite athletes suggest they may be more aware of the potential financial and nonfinancial benefits of such a win, and/or less optimistic about their potential to move up in the level of competition without the use of PEDs.

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.

The use of performance-enhancing drugs is a significant problem in sport. It cheats clean athletes of their hard-earned rewards from perfecting their skills though dedication and hard work. It defrauds fans by substituting a distorted playing field for a true competition. Anti-doping agencies have been charged with enforcing drug policies, primarily through the use of drug testing programs. We propose that drug testing, while important, is not sufficient to achieve deterrence. Engaging the principles of perceptual deterrence and development of a moral community of athletes can greatly enhance compliance with rules.

ObjectiveThe purpose of this narrative review was to summarize available data on testosterone levels in normal, healthy adult males and females, to provide a physiologic reference framework to evaluate testosterone levels reported in males and females with conditions that elevate androgens, such as disorders of sex development (DSD), and to determine the separation or overlap of testosterone levels between normal and affected males and females.MethodsA literature review was conducted for published papers, from peer reviewed journals, reporting testosterone levels in healthy males and females, males with 46XY DSD, and females with hyperandrogenism due to polycystic ovary syndrome (PCOS). Papers were selected that had adequate characterization of participants, and description of the methodology for measurement of serum testosterone and reporting of results.ResultsIn the healthy, normal males and females, there was a clear bimodal distribution of testosterone levels, with the lower end of the male range being four‐ to fivefold higher than the upper end of the female range(males 8.8‐30.9 nmol/L, females 0.4‐2.0 nmol/L). Individuals with 46XY DSD, specifically those with 5‐alpha reductase deficiency, type 2 and androgen insensitivity syndrome testosterone levels that were within normal male range. Females with PCOS or congenital adrenal hyperplasia were above the normal female range but still below the normal male range.ConclusionsExisting studies strongly support a bimodal distribution of serum testosterone levels in females compared to males. These data should be considered in the discussion of female competition eligibility in individuals with possible DSD or hyperandrogenism.

The most important element in achieving athlete compliance with anti-doping rules is the certainty of detection. Thus, scientific research plays a mission critical role in achieving clean competition. Many factors contribute to the advances in detection. Incremental advances in the ability to detect prohibited substances and methods, and identification of long-lived metabolites continue to lengthen detection windows. While the athlete biological passport hematological and steroidal modules hold great promise, experience shows that new research is needed to improve the sensitivity and specificity of the approach for current doping techniques. Indirect detection strategies using biomarkers or transcriptomic techniques have been increasingly investigated. The incorporation of more cost-effective sampling strategies using dried blood and plasma spots, oral fluid, and breath analysis show great promise toward increasing the number of tests while remaining within testing budget constraints. Despite the importance of research to ensuring rule compliance, a major challenge for anti-doping research is achieving and maintaining sufficient funding in the reality of the myriad of new substances introduced for disease treatment but abused for performance enhancement. In addition, obtaining metabolism and population reference range data, particularly for new drugs or designer drugs that have not obtained approval for administration to human subjects, remains a significant problem. Nevertheless, research continues to contribute important data to support anti-doping efforts.

The most important element in achieving athlete compliance with anti-doping rules is the certainty of detection. Thus, scientific research plays a mission critical role in achieving clean competition. Many factors contribute to the advances in detection. Incremental advances in the ability to detect prohibited substances and methods, and identification of long-lived metabolites continue to lengthen detection windows. While the athlete biological passport hematological and steroidal modules hold great promise, experience shows that new research is needed to improve the sensitivity and specificity of the approach for current doping techniques. Indirect detection strategies using biomarkers or transcriptomic techniques have been increasingly investigated. The incorporation of more cost-effective sampling strategies using dried blood and plasma spots, oral fluid, and breath analysis show great promise toward increasing the number of tests while remaining within testing budget constraints. Despite the importance of research to ensuring rule compliance, a major challenge for anti-doping research is achieving and maintaining sufficient funding in the reality of the myriad of new substances introduced for disease treatment but abused for performance enhancement. In addition, obtaining metabolism and population reference range data, particularly for new drugs or designer drugs that have not obtained approval for administration to human subjects, remains a significant problem. Nevertheless, research continues to contribute important data to support anti-doping efforts.

Despite the high prevalence of performance-enhancing drug (PED) use, media attention has focused almost entirely on PED use by elite athletes to illicitly gain a competitive advantage in sports, and not on the health risks of PEDs. There is a widespread misperception that PED use is safe or that adverse effects are manageable. In reality, the vast majority of PED users are not athletes but rather nonathlete weightlifters, and the adverse health effects of PED use are greatly underappreciated. This scientific statement synthesizes available information on the medical consequences of PED use, identifies gaps in knowledge, and aims to focus the attention of the medical community and policymakers on PED use as an important public health problem. PED users frequently consume highly supraphysiologic doses of PEDs, combine them with other PEDs and/or other classical drugs of abuse, and display additional associated risk factors. PED use has been linked to an increased risk of death and a wide variety of cardiovascular, psychiatric, metabolic, endocrine, neurologic, infectious, hepatic, renal, and musculoskeletal disorders. Because randomized trials cannot ethically duplicate the large doses of PEDs and the many factors associated with PED use, we need observational studies to collect valid outcome data on the health risks associated with PEDs. In addition, we need studies regarding the prevalence of PED use, the mechanisms by which PEDs exert their adverse health effects, and the interactive effects of PEDs with sports injuries and other high-risk behaviors. We also need randomized trials to assess therapeutic interventions for treating the adverse effects of PEDs, such as the anabolic-androgen steroid withdrawal syndrome. Finally, we need to raise public awareness of the serious health consequences of PEDs.

This summer, as the XXX modern Olympic Games and the XIV Summer Paralympic Games were held in London, millions watched magnificent efforts from the best athletes in the world. The ability of the athletes to produce their best personal performances in the spotlight and the pressure of the Olympic Games was a testament to the human spirit. But, because the margin between winning and losing is so small, one imprecisely placed foot or hand, one moment of indecision, one gust of wind at the wrong moment, and the gold medal is gone. Behind the scenes at these Olympics—properly so— was a scientific team performing under great pressure to ensure safe and fair competition by testing urine and blood for traces of performance-enhancing prohibited substances and methods. Their efforts were crucial, because, in partnership with the clean athletes, they support the integrity and the spirit of sport. More than 1000 samples were analyzed within a few days after each event for stimulants, steroids, masking agents, recombinant proteins like erythropoietin and growth hormone (GH), and other substances on the World AntiDoping Agency (WADA) Prohibited List (1). The List of Prohibited Substances and Methods is maintained by a committee of internationally recognized scientists and sport administrators. A revised list is released annually by the WADA. In deciding whether to add a compound or method to the list, the committee considers whether there is potential to enhance performance; a potential health risk for the athlete; or if use of the substance or method violates the spirit of sport. If two of the three criteria are met, the substance is added to the Prohibited List. The inclusion of the word “potential” recognizes that a substance may emerge for which there are no toxicological data. For example, the identification of the “designer” steroid tetrahydrogestrinone (THG) in a drop of fluid in a syringe turned in to the U.S. Anti-Doping Agency (USADA) in 2003 illustrates the problem. THG had been developed by Wyeth in the 1960s and taken to clinical trials as a potential anabolic agent, but never brought to market. It was impossible to locate any records from the clinical trials 40 years after the fact. THG had been shown to have anabolic activity (2). There were no publications about toxicological studies, but as a 17 -methyl steroid, comparison to similar structures strongly suggested the potential for some degree of hepatotoxicity. Based on this information, THG was added to the Prohibited List in 2006.

Methylhexaneamine (MHA) has been marketed in dietary supplements based on arguments that it is a constituent of geranium (Pelargonium graveolens) leaves, stems, roots or oil, and therefore qualifies as a dietary ingredient. The purpose of this study is to determine whether P. graveolens plant material (authenticated) or its oil contains detectable quantities of MHA. Two analytical methods were developed for the analysis of MHA in P. graveolens using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. The results were further confirmed using liquid chromatography-high-resolution mass spectrometry. Twenty commercial volatile oils, three authenticated volatile oils and authenticated P. graveolens leaves and stems (young and mature, and fresh and dried) were analyzed for MHA content. In addition, three dietary supplements containing MHA that alleged P. graveolens as the source are analyzed for their MHA content. The data show that none of the authenticated P. graveolens essential oils or plant material, nor any commercial volatile oil of Pelargonium (geranium oil) contain MHA at detectable levels (limit of detection: 10 ppb). The dietary supplements that contained MHA as one of their ingredients (allegedly from geranium or geranium stems) contained large amounts of MHA. The amounts of MHA measured are incompatible with the use of reasonable amounts of P. graveolens extract or concentrate, suggesting that MHA was of synthetic origin.

Blackledge has reviewed a portion of the instrumental data in the Landis doping arbitration case. This rebuttal to his review presents an impartial review of the gas chromatography-combustion-isotope ratio mass spectrometric data in the case. The rebuttal also discusses the responsibilities of expert witnesses in courtroom testimony.

The detection and deterrence of the abuse of performance-enhancing drugs in sport are important to maintaining a level playing field among athletes and to decreasing the risk to athletes’ health. The World Anti-Doping Program consists of six documents, three of which play a role in analytical development: The World Anti-Doping Code, The List of Prohibited Substances and Methods, and The International Standard for Laboratories. Among the classes of prohibited substances, three have given rise to the most recent analytical developments in the field: anabolic agents; peptide and protein hormones; and methods to increase oxygen delivery to the tissues, including recombinant erythropoietin. Methods for anabolic agents, including designer steroids, have been enhanced through the use of liquid chromatography/tandem mass spectrometry and gas chromatography/combustion/isotope-ratio mass spectrometry. Protein and peptide identification and quantification have benefited from advances in liquid chromatography/tandem mass spectrometry. Incorporation of techniques such as flow cytometry and isoelectric focusing have supported the detection of blood doping.

Doping control for national- and international-level athletes has undergone major changes in the past few years, and will continue to change at an accelerated rate. National antidoping organizations (NADOs) such as the United States Anti-Doping Agency (USADA) are being established by major nations to work with national governing bodies of sport. The World Anti-Doping Agency has been established to coordinate worldwide antidoping efforts with the NADOs and international federations of sport, and to implement a recently drafted World Anti-Doping code, which clarifies the definition of doping and establishes procedures to harmonize international efforts in sample collection process, testing laboratory accreditation, result reporting, and result adjudication. A number of substances and methods currently used in doping present serious challenges to the scientific community, and are described briefly. In addition, brief descriptions of other issues of significance to doping control, including the role of physicians in doping and the operation of the USADA, are presented.

The use of stimulants such as amphetamine, cocaine, strychnine, and ephedrine in modern sport to improve focus and delay fatigue was reported in the early 1900's. In response to the death of a Danish cyclist at the 1960 Olympics, the death of a UK cyclist at the Tour de France, and other evidenc

Can we catch every person violating anti-doping rules through just a random drug test given the current resources? No. The police cannot catch every impaired driver, so should they stop trying? Regulators cannot catch every incidence of insider trading on Wall Street, so should we cease regulation of the finance industry? And should we stop performing Internal Revenue Service audits because some people successfully cheat on their taxes? The fundamental problem with the question posed based on a Bayesian analysis of testing numbers is that it assumes that catching every cheater through random drug testing is the sole purpose of an antidoping program. It is not. But before focusing on the role of drug testing in an antidoping program, why are antidoping rules important? To live together and achieve societal goals, we agree to a set of rules to govern our work and play. We accept rules against impaired driving, for example, because of the potential harm to others. We accept rules that the basketball rim is 3.05 m high and the court is 28 by 15 m to have a level playing field. Violating the rules that the athlete agrees to abide by to have equitable competition has its own name: cheating. When an individual cheats, whether in sport, at work, or on their taxes, it defrauds or takes something away from others. It is ethically and morally wrong, no matter how the individual tries to rationalize it. Cheating by violating antidoping rules is not a victimless crime. Cyclist Scott Mercier was forced to make the choice to quit the sport he loved because he was unwilling to become a fraud and risk his health. Shotputter Adam Nelson recently received his gold medal 9 years after the 2004 Olympic contest because reanalysis of the samples with new technology revealed …

Detection of performance-enhancing drugs in sports has received increasing visibility. Athletic drug testing uses sophisticated technology and both interindividual (population) and intraindividual reference ranges to interpret data. An effective program must incorporate educational and adjudication components in addition to testing. The difficult interface between science and the law is evident in many recent sports arbitration decisions.

Measurement of the ratio of testosterone (T) and epitestosterone (E) in urine has been used as an indication of 'natural' steroid supplementation for a decade. The direct measurement of the glucuronide and sulfate conjugates of testosterone and epitestosterone by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) should resolve a number of issues regarding unusual metabolism due to either genetic disposition or attempts to avoid detection of abuse. Determination of nanomoles per liter (0.1 ppb) concentrations of analytes in a complex biological matrix by HPLC/MS/MS is complicated by sample matrix-specific ion suppression during ESI. Deuterated internal standards of all compounds were used to overcome the effects of suppression. Comparison of the HPLC/MS/MS method with a two-part gas chromatographic/mass spectrometric method showed statistical equivalence in urine samples. Analysis of urine samples with elevated T-glucuronide to E-glucuronide ratios did not show that a significant number could be explained by an elevated excretion of epitestosterone sulfate. The HPLC/MS/MS method was also used further to characterize genetic and metabolic factors that give rise to unusual T/E ratios.

The use of drugs to enhance performance in sports has certainly occurred since the time of the original Olympic Games. The origin of the word “doping” is attributed to the Dutch word “doop,” which is a viscous opium juice, the drug of choice of the ancient Greeks. The use of stimulants, such as amphetamine, cocaine, strychnine, and ephedrine, in sports was reported in the early 1900s. It is interesting to note that a 1997 survey of student-athletes indicated an increased use of ephedrine. Of the small number of respondents who indicated use of ephedrine, 50% indicated an intent to improve performance. 28 In response to the death of a Danish cyclist at the 1960 Olympics and other evidence of widespread use of potentially life-threatening drugs in sports, the Council of Europe (COE) established the following definition of doping in 1963: The administering or use of substances in any form alien to the body or of physiological substances in abnormal amounts and with abnormal methods by healthy persons with the exclusive aim of attaining an artificial and unfair increase in performance in competition. Furthermore, various psychological measures to increase performance in sports must be regarded as doping. Where treatment with a medicine must be undergone, which as a result of its nature or dosage is capable of raising physiological capability beyond normal level, such treatment must be considered doping and shall rule out eligibility for competition. It should be noted that the most recent known death owing to the use of a drug for performance enhancement was in 1993 when a body builder died of a heart attack caused by diuretic-induced hypokalemia. Having established the basis for doping in athletics, an initial list of banned substances was published by the COE, which included narcotics, amine stimulants, alkaloids, analeptic agents, respiratory tonics, and certain hormones. The International Olympic Committee (IOC) established a Medical Commission in 1967 charged with various responsibilities in the prohibition of doping, including the maintenance of a list of prohibited substances and methods (vide infra). The IOC also adopted a Medical Code “to protect the health of athletes and to ensure respect for the ethical concepts implicit in Fair Play, the Olympic Spirit, and medical practice.” 33 Other national and international sport-governing bodies, especially in Europe, quickly established rules regarding the use of drugs. Interest in doping in North America was stimulated by Congressional hearings in 1973 on the improper use of drugs in sports in the United States and the 1988 Dubin Commission inquiry in Canada. 19 Major athletic organizations in the United States, including the National Collegiate Athletic Association (NCAA), the National Football League (NFL), USA Track and Field (USATF; formerly The Athletics Congress), and the United States Olympic Committee (USOC), implemented comprehensive drug programs during this period. Both educational efforts and testing for prohibited substances are an integral part of all of these programs. In recent years, the line between training for a competitive advantage and the “pharmacologic advantage” explicitly banned in the COE definition of doping has become more difficult to discern. The use of specially researched and prepared diets, including supplements of amino acids, creatine, vitamins, and minerals, to achieve training goals might seem a small step from taking a “natural” supplement that contains ephedrine, strychnine, or androstenedione. Modification of the training environment (e.g., adjusting the oxygen concentration) might seem analogous to administration of recombinant erythropoietin (EPO). Athletes justify bending the rules for a number of reasons. By his or her nature, the elite athlete is driven to succeed. Most athletes surveyed have indicated in an anonymous survey their willingness to take prohibited substances if they could not be caught and would consistently win. 4 Violating the rules and forcing officials to either call the infraction and impose a penalty or accept the illegal behavior can be observed at any level of competition, and in some cases is coached. The second arises from the fact that if the athletes perceive that the competition is winning because of the use of performance-enhancing drugs, they must use them also. A recent NCAA survey indicated that the athletes, at least in this area of competition, believe that the drug control program works in preventing use. This, in turn, means that everyone can compete drug-free. The athlete is also surrounded by individuals committed to their success. As has become clear from the exposition of the German Democratic Republic doping program, 21 at times the athlete needs to be protected from those around them. Although the imposition of testing and strict liability for what is a controversial issue, it is central to controlling drug use. The deterrent effect of testing is an important part of its value. In the end, however, it is the ethics of the athlete and their entourage that determine whether or not to succomb to the lure of drugs.

The practice of therapeutic drug monitoring is based on several disciplines, including pharmacokinetics, pharmacodynamics, and chemical analysis. The impact of the analysis on the determination of pharmacokinetic parameters is not well appreciated. Analytical goals in therapeutic drug monitoring should be established by determining the nature of the problem to be solved, selecting the appropriate matrix and methodology to solve the problem, and developing valid analytical schemes that are performed competently with appropriate quality and interpreted within the framework of the problem.

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG β-subunit. After reductive alkylation of the nicked hCG β-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked β-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.

The use and abuse of performance-enhancing substances has been an issue in sports since the ancient Greeks. The availability of numerous synthetic steroids and recombinant peptide hormones has made testing an analytical challenge. Recent advances in mass spectrometry have provided an opportunity to decrease detection limits. The Atlanta Olympic Games in 1996 marked the first time every specimen was screened by gas chromatography (GC) coupled to high-resolution mass spectrometry (MS). A further improvement may be seen with GC/MS/MS and quadrupole ion traps. Electrospray HPLC/MS has also been applied to the detection and confirmation of peptide hormones in urine. The ability to detect subtle differences in oligosaccharide structure may provide a way to detect abuse of recombinant glycoproteins. Simply decreasing detection limits is not enough; new technology also allows development of a foundation on which to base interpretation. Application of HPLC/MS/MS has allowed direct measurement of steroid conjugates in urine. The relative importance of sulfate, glucuronide, and other conjugates and metabolites of testosterone and epitestosterone can now be assessed. In the international sports arena, the impact of genetic metabolic disposition must also be considered if we are to provide an equitable system. Further research will establish more-refined criteria for the detection threshold of abused substances.

Limits of detection are important issues for GC/MS screening for anabolic agents and for confirmation of various drugs of abuse. We compared a quadrupole ion trap (QIT) operated in two different selected-ion storage modes and a quadrupole mass filter (QMF) operated in the selected-ion monitoring mode. Results with the model compound tetrachlorobenzene indicate that, for simultaneous monitoring of more than four ions, the QIT operated in a frequency-modulated selected-ion storage mode has better limits of detection than the QMF. Use of a single-ion storage technique gave results similar to those of the QMF. We also evaluated both QIT selected-ion storage approaches for the limits of detection of the trimethylsilyl derivatives of four anabolic steroid metabolites and the β-agonist clenbuterol. We found no improvement in detection limits over that of a similar method with selected-ion monitoring and a QMF when four anabolic steroid metabolites and clenbuterol were extracted from a urine matrix. The lack of improvement in the limit of detection resulted from matrix background signals at masses similar to those of the steroids.

A high-performance liquid chromatographic/electrospray mass spectrometric (HPLC/MS) technique is described for the characterization of the β-subunit of the glycopeptide human chorionic gonadotropin (hCG). The β-subunit of hCG was dissociated from the α-subunit using 0.1% trifluoroacetic acid (TFA) and separated by reversed-phase HPLC using a 0.1% TFA–acetonitrile gradient. Although reductive alkylation with 4-vinylpyridine allowed direct observation of the intact β-subunit of hCG by HPLC/MS due to the increase in charge, the heterogeneity of the carbohydrate fractions resulted in poor detection limits and extremely complex spectra. After reductive alkylation with either iodoacetate or 4-vinylpyridine, tryptic fragments of either the α- or β-subunit can be observed using reversed-phase HPLC/MS. HPLC/MS data were consistent with the reported primary sequence, although oligosaccharide attachment sites at both 127Ser and 132Ser could not be documented. Microheterogeneity of the carbohydrate moiety on both N-glycosylation sites on the β-subunit could be readily observed. A larger degree of heterogeneity was observed on 13Asn. Differences were also observed in the oligosaccharide distribution in three commerial preparations of hCG. Detection of the C-terminal portion of the β-subunit required enzymatic deglycosylation prior to HPLC/MS analysis. © 1997 by John Wiley & Sons, Ltd.

BACKGROUND Insulin-like growth factor 1 (IGF-1)7 is a key mediator of growth hormone (GH) action and a well-characterized biomarker of GH abuse. Current immunoassays for IGF-1 suffer from poor concordance between platforms, which makes comparison of results between laboratories difficult. Although previous work has demonstrated good interlaboratory imprecision of LC-MS/MS methods when plasma is supplemented with purified proteins, the interlaboratory imprecision of an endogenous protein in the nanogram-per-milliliter concentration range has not been reported. METHODS We deployed an LC-MS/MS method to quantify serum IGF-1 in 5 laboratories using 5 different instruments and analyzed 130 healthy human samples and 22 samples from patients with acromegaly. We determined measurement imprecision (CV) for differences due to instrumentation, calibration curve construction, method of calibration, and reference material. RESULTS Instrument-dependent variation, exclusive of digestion, across 5 different instrument platforms was determined to be 5.6%. Interlaboratory variation was strongly dependent on calibration. Calibration materials from a single laboratory resulted in less variation than materials made in individual laboratories (CV 5.2% vs 12.8%, respectively). The mean imprecision for 152 samples between the 5 laboratories was 16.0% when a calibration curve was made in each laboratory and 11.1% when a single-point calibration approach was used. CONCLUSIONS The interlaboratory imprecision of serum IGF-1 concentrations is acceptable for use of the assay in antidoping laboratories and in standardizing results across clinical laboratories. The primary source of variability is not derived from the sample preparation but from the method of calibration.

A method has been developed for confirmation of the glycopeptide human chorionic gonadotropin (hCG) in urine. A solid-phase immunoaffinity trapping technique utilizing a monoclonal antibody recognizing both intact hCG and free hCG beta-subunits was developed for the extraction of hCG from urine. Recovery of hCG from a urine matrix was essentially quantitative. The hCG was quantitatively eluted with 6 M guanidine hydrochloride, reductively alkylated with vinylpyridine, and subjected to tryptic digestion. The tryptic digest was analyzed by HPLC-MS. Ions from three tryptic fragments were monitored with selected ion monitoring to provide specific detection of hCG. The signal observed for a concentration of 25 mIU/ml of hCG could be clearly distinguished from background with a signal-to-noise ratio of 12:1.

Cyclosporin A (CsA) given to Sprague-Dawley rats in vivo produced a tissue-specific, dose-dependent inhibition of translation elongation in renal microsomes. CsA at an oral dose of 50 mg/kg/day for 6 days reduced renal microsomal translation by 70.5%. Renal cytoplasm from rats treated in vivo with CsA inhibited translation by 55% when added to renal microsomes isolated from tissues of control animals. In contrast, CsA added to renal microsomes in vitro did not inhibit translation. Renal cytoplasm from CsA-treated rats containing translation inhibitory factor was found by HPLC to contain CsA and CsA metabolites M1 and M17. CsA metabolites M1, M17, M18 and M21 were isolated from human bile and tested in vitro for translation elongation inhibitory activity in renal microsomes. CsA, M18 and M21 did not inhibit translation elongation at concentrations of up to 2500 ng/ml. M17 inhibited translation elongation, but only by 8.4% at the highest concentration tested (2500 ng/ml), a level 20-fold higher than that measured in renal cytoplasm (125 ng/ml). Ml produced a concentration-dependent inhibition of translation elongation, beginning at 500 ng/ml, or approximately 2-fold higher than that found in renal cytoplasm (260 ng/ml). M1 at 2500 ng/ml or approximately 10-fold higher than the concentration measured in renal cytoplasm, inhibited translation elongation by 23.8%, only 1 3 that observed upon addition of renal cytoplasm containing translation inhibitory factor. We conclude from these findings that the dose-dependent inhibition of renal translation elongation following in vivo CsA cannot be explained by the renal formation or uptake of known CsA metabolites.

The practice of using prohibited substances to enhance sports performance (typically referred to as “doping”) is a very old practice that has been with us for centuries. To ensure harmonized, coordinated, and effective antidoping programs, the World Anti-Doping Agency (WADA)6 was created as an independent agency. The WADA publishes a list of prohibited substances annually, and the presence of any prohibited substance or metabolite in an athlete's sample constitutes an antidoping rule violation. Over the years, testing programs have continued to expand, and WADA-accredited laboratory testing has increased 40% over the last 3 years. The issue of sports doping has gained widespread attention, and the public seems to be continually reminded of athletes who dope and the potential growing problem of doping at all levels of competition. Surprisingly, an athlete has come forward and stated that it is impossible to win a cycling event like the Tour de France without doping. In this Q&A article, 4 leaders with different roles in the antidoping effort have been asked to comment on the success of antidoping programs and major challenges facing antidoping programs, elite athletes, and laboratories in the fight to maintain fair and clean competition. According to WADA statistics, only a small percentage of athletes who are tested test positive for prohibited substances. Do you believe that the use of prohibited substances is more widespread? John A. Lombardo: Establishing the prevalence of prohibited-substances use by results of drug testing would be similar to establishing the prevalence of driving under the influence of alcohol by the number of arrests for drinking and driving. A common goal of all drug-testing programs is to level the playing field so that athletes do not feel they have to use performance-enhancing drugs or prohibited substances to be competitive. Drug-testing programs serve the dual purpose of …

The fight against the use of performance-enhancing drugs in sports has been in effect for nearly 90 years. The formation of the World Anti-Doping Agency in 1999 was a major event because an independent agency was entrusted with harmonization of the antidoping program. In addition to sports governing bodies, governments have endorsed WADA and its programs by signing a United Nations Education, Science, and Cultural Organization Convention on Doping. The first step in the harmonization process was the development of the World Anti-Doping Program. This program consisted of five documents - the Code, the International Standard for Testing, the International Standard for Laboratories, the Prohibited List, and the International Standard for Therapeutic Use Exemptions - which unified the approach of the international federations and national antidoping agencies in applying antidoping rules. For laboratory testing, the International Standard for Laboratories establishes the performance expectations for and competence of laboratories recognized by WADA, including accreditation under ISO/IEC 17025. The antidoping rules are adjudicated by arbitration using the internationally recognized Court of Arbitration for Sport.

In the days leading up to the 2008 Olympic Games in Beijing, a number of scientific journals published special issues (1)(2) or critical commentaries and opinion pieces regarding the antidoping movement. For example, an analysis of antidoping policy by Kayser and Smith (3) presented criticisms based on ethical arguments (4). On the other hand, equally persuasive ethical arguments supporting antidoping policies were reported by Murray (5). In addition, the public statements released by the defense team before each of the 2 arbitration hearings for Tour De France winner Floyd Landis, at which he was convicted of committing a doping violation, vehemently attacked the laboratory and the staff that performed the test. These public attacks were by advocates for the defendant, and the antidoping rules prevented a response to these charges. My purpose here is to provide an overview of how the current antidoping rules came into effect, to review some of the science underlying the antidoping rules, and to discuss a few of the cases decided by the Court of Arbitration in Sport (CAS).1 The fight against performance-enhancing substances began in the 1920s, long before there were testing methods for the prohibited substances. For years, the leaders of sport went it alone in this fight. Some critics argued, and continue to argue, that sport leaders have an issue similar to the “fox guarding the henhouse” when it comes to overseeing drug-testing programs. A major shift occurred in 1999, when the World Anti-Doping Agency (WADA) was formed as an independent body to harmonize antidoping practices, and independent national antidoping organizations such as the Australian Sport Drug Agency, the Canadian Center for Ethics in Sport, and the United States Anti-Doping Agency (USADA) began to emerge. WADA was established as a Swiss foundation with the support of intergovernmental bodies, governments, …

The fight against the use of performance-enhancing drugs in sports has been in effect for nearly 90 years. The formation of the World Anti-Doping Agency in 1999 was a major event because an independent agency was entrusted with harmonization of the antidoping program. In addition to sports governing bodies, governments have endorsed WADA and its programs by signing a United Nations Education, Science, and Cultural Organization Convention on Doping. The first step in the harmonization process was the development of the World Anti-Doping Program. This program consisted of five documents – the Code, the International Standard for Testing, the International Standard for Laboratories, the Prohibited List, and the International Standard for Therapeutic Use Exemptions – which unified the approach of the international federations and national antidoping agencies in applying antidoping rules. For laboratory testing, the International Standard for Laboratories establishes the performance expectations for and competence of laboratories recognized by WADA, including accreditation under ISO/IEC 17025. The antidoping rules are adjudicated by arbitration using the internationally recognized Court of Arbitration for Sport.

We studied dietary branched-chain amino acid enrichment in cholestatic weanling rats. Growth was assessed with body weight, muscle weight and nitrogen balance. Systemic metabolic measurements that reflect liver function were evaluated, including plasma ammonia, albumin, amino acids, glucose, triglyceride and branched-chain ketoacids, as well as urinary carnitine excretion. Twenty-two rats underwent bile-duct ligation at 14 d of age. At weaning, 11 rats were fed a control diet and 11 an isoenergetic, isonitrogenous branched-chain amino acid-enriched diet for 3 wk, each with a sham-operated, pair-fed control. Body weights were similar in all four groups. Changes due to bile-duct ligation and not affected by the diet manipulation included lower plasma glucose, nitrogen balance and muscle weight, and higher triglyceride concentration, carnitine excretion and liver weight. Changes due to ligation that were normalized by dietary manipulation included plasma albumin, ammonia and total amino acid concentrations. The ratio of branched-chain to aromatic amino acids was decreased in ligated animals fed both diets; however, branched-chain amino acids were lower in the two groups fed more branched-chain amino acids.

The use of quantitative cocaine urinalysis is illustrated with case reports from four subjects. Urine samples were collected for a period of 21 consecutive days and analyzed using fluorescent polarization immunoassay. Urinalysis results were compared to self-reported use. Findings support the use of quantitative urinalysis as an outcome measure in clinical research. Self-reported high-dose use was associated with higher urine concentrations and more protracted periods of decline. The importance of repeated urinalysis is discussed.

Therapeutic monitoring of cyclosporine has been a controversial area. The unique physiological and pharmacological properties of cyclosporine are partly responsible for the inconsistent correlation between blood concentrations and clinical effect. The variety of analytical methods has also contributed to conflicting opinions. Recently, a number of selective methods for cyclosporine in whole blood have become available, which should help produce consistent results between laboratories. The value of therapeutic monitoring appears to be decreasing the incidence of nephrotoxicity and identifying noncompliance.

On the basis of cocaine-caused kindling in animals and the usefulness of carbamazepine in treating kindling-type seizures, carbamazepine has been tried in clinical settings with cocaine-dependent individuals. This report presents findings of a 20-day, double-blind, placebo-controlled crossover study in 32 nontreatment-motivated, paid, chronic crack cocaine users. Carbamazepine significantly lowered the mean number of positive urine specimens compared with placebo. Of clinical importance, serum carbamazepine levels of 4 micrograms/ml (17 mumol/L) or more were associated with greater improvement. A consistent, clinically important trend linked therapeutic levels with improvement for all subjective and objective outcome variables. Comparison of daily acknowledged cocaine use or professed cocaine abstinence, with cocaine use indicated by daily urinalysis in these chronic cocaine users, has suggested the possibility of cocaine saturation as an important methodologic limitation inherent in outpatient studies of cocaine use in humans.

We report a micromethod for the analysis of cyclosporine (CsA), based on quantification of its constituent amino acids. The amino acids were released by gas-phase hydrolysis, derivatized with fluorenylmethyl chloroformate, and separated and analyzed in a reversed-phase HPLC system. The imprecision (CV) of the amino acid analysis was less than 4%, and several determinations of the amount of standard CsA were within 1% of the weighed material. The detection limits (signal-to-noise ratio = 2) were 500 fmol for ultraviolet detection and 100 fmol for fluorescence detection. We also used this method to determine the ultraviolet absorptivities of CsA and five metabolites at 210, 214, and 230 nm. The molar absorptivity of most metabolites was about 10% higher than that of CsA, although the metabolite that was oxidized to a carboxyl group on the terminal carbon of N-methyl-butenyl-methyl-threonine (AM1A) had a molar absorptivity about 40% higher than that of CsA.

Despite the fact that cyclosporine (CsA) has been used clinically for a number of years, there is still uncertainty about the efficacy of monitoring its blood levels. Few if any studies have documented clear differentiation of rejection, immunosuppression, and toxicity on the basis of CsA concentrations alone. The issues in CsA monitoring include selection of sample matrix, analytical method, dosing interval and the timing of trough measurements, the temporal relationship between measurements and physiological events such as toxicity, the concurrent presence of multiple other immunosuppressive agents, and the lack of "gold standards" for determining rejection, adequate immunosuppression, and toxicity. In contrast to using CsA levels for the differential diagnosis of rejection and toxicity, there is evidence that maintenance of CsA concentrations within a therapeutic window results in a lower prevalence of toxicity and rejection.

The availability of endogenous anabolic steroids and their precursors in the form of “dietary supplements” has become widespread in the United States. The popularity of dehydroepiandrosterone (DHEA) arose from a number of reports in the popular press that suggested that the results of a study conducted by Morales et al. (1) characterized DHEA as similar to the mythical fountain of youth. Increased use of DHEA among athletes was reported anecdotally before and during the 1996 Olympic Games, and the International Olympic Committee (IOC) Medical Commission explicitly added the compound to the list of prohibited compounds in December 1996, although it would have been considered prohibited previously under the “related compounds” provision of the list. Other groups testing for steroids have either added DHEA as an example of a prohibited compound or assumed that it did not play a role in their testing program. Some researchers have questioned whether the use of compounds that are precursors of the anabolic steroid testosterone increase testosterone and therefore impact the testosterone/epitestosterone (T/E) ratio. Two studies have reported that no increase in the T/E ratio occurred when DHEA was given (2)(3). We report here on the impact of administration of two over-the-counter DHEA preparations on the excretion of several steroids, and a greater than 6:1 dose-dependent increase of the T/E ratio in one individual. Two over-the-counter preparations of DHEA were obtained from a health food store and from a pharmacy. Nature’s Pride “DHEA 50 mg+” (product A; Nature’s Products, Davie, FL) capsules contained DHEA (50 mg), suma ( Pfaffia paniculata , 25 mg), Korean ginseng ( Panax ginseng , 25 mg), muira pauma ( Ptychopetalum olacoides , 25 mg), shitake mushroom concentrate ( Lentinus edodes , 15 mg), and green tea extract ( Camelia sinesis , 5 mg). YourLife DHEA tablets (product B; Leiner Health Products, Inc., Carson, …

The first documented use of testosterone as a performanceenhancing substance in sport began in the 1950s. The success of Russian weightlifters during this era led Dr. John B. Zeigler to surreptitiously obtain information from their trainers that they were using testosterone. Later, Zeigler would use Dianabol to enhance the performance of weightlifters at the York Barbell Club beginning in 1958. Once Pandora’s Box was open, it was impossible to close. The growth and widespread use of anabolic steroids in many competitive sports eventually led the U.S. Congress to schedule testosterone and other anabolic agents under the Anabolic Steroids Control Act in 1990 (Public Law 101647). For many years, the medical community argued that there was no proof that testosterone enhanced athletic performance. Finally, in 1996, Bhasin and co-workers showed that supraphysiological doses of testosterone indeed produced performance benefits (1), something the athletes had known for four decades. The urinary testosterone to epitestosterone (T/E) ratio was the first test to be used for evaluation of abuse of a natural (or endogenous) substance such as testosterone to enhance performance in sport. The production of reliable quadrupole gas chromatograph/mass spectrometers in the mid-1970s allowed more widespread study of urinary steroid profiles. Donike and coworkers (2) observed an unusual distribution of testosterone concentrations in samples collected at the Moscow Olympics in 1980. In 1982, Donike proposed the use of the urinary T/E ratio based on the observation that epitestosterone was excreted in the urine in a relatively constant amount and as such constituted a way to compensate for the variability in testosterone concentration that occurred as a function of the diluteness of the urine (2). Because the primary metabolite of testosterone is testosterone glucuronide, the procedure developed by Donike used -glucuronidase in the preparation of the samples for analysis. In 1983, the International Olympic Committee accepted a T/E ratio of greater than 6:1 as evidence of testosterone doping, based on the 99th percentile of the log normal distribution of ratio values established by Donike and his co-workers. It was soon clear that the T/E ratio based on population reference ranges had limitations. For example, it had been noted in 1970 that genetic make-up could have a significant effect on urinary testosterone excretion, whereas epitestosterone excretion was unaffected (3). As population T/E ratio data accumulated in the anti-doping laboratories, it was clear that there were two distributions of urinary T/E ratios in both men and women, one with a maximum frequency at a T/E ratio of about 0.1 and a much larger population with a maximum frequency at a T/E ratio of about 1. Within the anti-doping community, the smaller population was referred to as low mode, and it was known that administration of testosterone to low-mode individuals did not result in a T/E ratio above the population threshold (4). Jakobsson and her colleagues (5) provided the mechanism for lowmode excretion in 2006 when they demonstrated that the UGT2B17 gene deletion was the cause of significant differences in testosterone glucuronide excretion between Korean and Swedish men. In their current paper in this issue, JakobssonSchulze et al. (6) report that when subjects deficient in the UGT2B17 gene (del/del) receive exogenous testosterone, their T/E ratio does not rise above the current population-based threshold of 4:1. One potential route of excretion of testosterone in UGT2B17 del/del genotypes is through other phase II metabolites such as testosterone sulfate. Borts and Bowers (7) directly measured the concentrations of testosterone and epitestosterone sulfate and glucuronide using HPLC/tandem mass spectrometry. Low-mode excretors did not produce larger than normal amounts of testosterone or epitestosterone sulfate. There was also no change in the relative production of sulfate conjugates. Because phase II metabolism does not explain the difference in testosterone handling in these individuals, it would be interesting to know what phase I metabolism products are increased in individuals with the UGT2B17 del/del polymorphism. This information may help develop a more sensitive test for testosterone

A metabolite of cyclosporine has been isolated and its structure identified through use of HPLC and tandem mass spectroscopy. Fast atom bombardment mass spectrometry of an HPLC fraction co-eluting with 1 eta hydroxy-cyclosporine (M17) indicated that the mass of this metabolite was 2 Da greater than that of cyclosporine. Further isolation by HPLC yielded a pure fraction, which we analyzed with tandem mass spectrometry. Linear acyl fragment ions originating from the metabolite under collision-induced dissociation were consistent with the difference in mass being associated with amino acid 9 in the cyclosporine backbone. We propose a nomenclature system for future discussion of cyclosporine metabolites.

The two most obvious differences between alkyl-bonded silica phases and polystyrene-divinylbenzene resins as reversed-phase chromatographic supports are the aromaticity and the lack of hydrogen bonding ability in the polymeric resin. The effect of these differences on the selectivity for a set of small solutes was studied through the use of a solvatochromic comparison method. For retention on a polymeric phase for mobile phases with the modifiers methanol and acetonitrile, the linear solvation energy relationship indicated an increased dependence on the polarizability/dipolarity of solutes. For the modifier tetrahydrofuran, retention on the polystyrene-divinylbenzen resin was indistinguishable from that on the alkyl-bonded silica phase. The hydrogen bonding ability of a solute was found to play a greater role in retention on alkyl-bonded silica than on the polymeric resin for all three modifiers. Since the mobile phase compositions were chosen such that the Hildebrand solubility parameters were equal, the dependence of retention on molar volume was found to be the same for all mobile phase-stationary phase combinations examined.

Gas chromatography-mass spectrometry (GC-MS) is the method of choice for separation and detection of anabolic steroids in urine. Recently, there have been advances in the areas of gas chromatography columns, tandem mass spectrometry using ion traps, and large volume sample injection that have promise for lowering detection limits and extending the utility of GC-MS for steroid analysis. In this work, a Varian Saturn III GC-MS system has been used in its tandem mass spectrometry mode to detect low picogram levels of model steroids in standard solution and the urine matrix. Application of MS-MS-MS provided structurally informative spectra for 3'-hydroxystanozolol at concentrations of 1 ng/ml. In addition, four polysilphenylene-polydimethylsiloxane capillary columns were examined for background and selectivity. The columns had bleed several-fold lower than conventional polysiloxane columns. The columns also exhibited significant differences in selectivity for structurally similar steroids. Finally, a new temperature-programmed split-splitless injector was used to inject as much as 25 microliters on column. The resulting limits of detection were 5 pg/ml for norandrosterone.

The analysis of trace components in complex biological matrices requires the use of reliable internal standards. For the gas chromatography/mass spectrometry (GC/MS) and high performance liquid chromatography/mass spectrometry (HPLC/MS) analyses, the stable isotope-labelled analogues of the analyte molecules are the most appropriate internal standards. In this work high-yield synthetic procedures for stably labelled and isotopically pure [16,16,17-2H3]-testosterone and- epitestosterone are reported. Synthetic methodologies for the glucuronidation and sulfation were established with the commercially available epitestosterone. Structure characterization of 4-androsten-17 alpha-ol-3-one methyl-2',3',4'-tri-O-acetyl-beta-D-glucuronate was made by two-dimensional nuclear magnetic resonance (COSY). Subsequently glucuronidation of [16,16,17-2H3]-testosterone and sulfation of [16,16,17-2H3]-epitestosterone were carried out at greater than 60% yield. However, the yield from the glucuronidation of epitestosterone was not as high. Electrospray mass spectrometry of four conjugates: testosterone sulfate, epitestosterone sulfate, testosterone glucuronide and epitestosterone glucuronide was carried out in the positive ion mode at a number of orifice voltages (50-95 V). Studies of the collisionally induced dissociation at both the interface and in the collision cell (MSMS) confirmed that the glycosidic bond of epitestosterone glucuronide was more labile than that of testosterone glucuronide. Use of the deuterated internal standards is reported to demonstrate the direct analysis of the steroid conjugates by HPLC/MS.

Cyclosporine (CsA) is extensively metabolized, with over 14 metabolites having been characterized to date. The confirmation of structure and purity is a prerequisite for studies involving CsA metabolites. Analytical techniques such as fast atom bombardment/mass spectroscopy (FAB/MS), tandem mass spectrometry (MS), 1H- and 13C-nuclear magnetic resonance (NMR) can be used for such purposes. In vitro experiments indicate that metabolites are considerably less immunosuppressive and toxic than CsA. In vivo studies have been hampered by sufficient quantities of metabolites and a suitable animal model. Preliminary results in the rat suggest that CsA metabolites are less immunosuppressive and toxic than CsA, although these results must be confirmed using a more suitable animal model. Present data indicate that the routine monitoring of metabolites is not warranted in transplant patients, although additional information is required to confirm these findings.