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.

Large divergence in testosterone concentrations between men and women: Frame of reference for elite athletes in sex-specific competition in sports, a narrative review.

OBJECTIVE: The 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. METHODS: A 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. RESULTS: In 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. CONCLUSIONS: Existing 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.

Achievements and Challenges in Anti-Doping Research.

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.

Treating the elite athlete: anti-doping information for the health professional.

Physicians and health professionals are a vital component in preserving the integrity of competition and the core principles of true sport. When treating an athlete, health professionals need to be cognizant of the anti-doping rules of the relevant sport organization. This review aims to provide an overview of the World Anti-Doping Agency Prohibited List, Therapeutic Use Exemptions, roles and responsibilities of the health professional, as well as provide resources that will guide their work with athletes.

Interlaboratory agreement of insulin-like growth factor 1 concentrations measured by mass spectrometry.

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.

Pelargonium oil and methyl hexaneamine (MHA): analytical approaches supporting the absence of MHA in authenticated Pelargonium graveolens plant material and oil.

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.

Science and the rules governing anti-doping violations.

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.

Advocacy versus impartial scientific review: a problem for scientists and the courts.

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 analytical chemistry of drug monitoring in athletes.

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.

An update on regulatory issues in antidoping programs in sport.

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.