Serological cancer-associated protein biomarker levels at bowel endoscopy: Increased risk of subsequent primary malignancy.

BACKGROUND: It was previously shown in three subpopulations that subjects not identified with colorectal cancer (CRC) at bowel endoscopy, but with increased serological cancer-associated protein biomarker levels had an increased risk of being diagnosed with subsequent malignant diseases. OBJECTIVE: The aim of the present study was to perform a pooled analysis of subjects from the three subpopulations and subsequently validate the results in an independent study. The study population denoted the training set includes N = 4,076 subjects with symptoms attributable to CRC and the independent validation set N = 3,774 similar subjects. METHODS: Levels of CEA, CA19-9, TIMP-1 and YKL-40 were determined in blood samples collected prior to diagnostic bowel endoscopy. Follow-up of subjects not diagnosed with CRC at endoscopy, was ten years and identified subjects diagnosed with primary intra- or extra-colonic malignant diseases. The primary analysis was time to a newly diagnosed malignant disease and was analyzed with death as a competing risk in the training set. Subjects with HNPCC or FAP were excluded. The cumulated incidence was estimated for each biomarker and in a multivariate model. The resulting model was then validated on the second study population. RESULTS: In the training set primary malignancies were identified in 515 (12.6%) of the 4,076 subjects, who had a colorectal endoscopy with non-malignant findings. In detail, 33 subjects were subsequently diagnosed with CRC and 482 subjects with various extra-colonic cancers. Multivariate additive analysis of the dichotomized biomarkers demonstrated that CEA (HR = 1.50, 95% CI:1.21-1.86, p < 0.001), CA19-9 (HR = 1.41, 95% CI:1.10-1.81, p = 0.007) and TIMP-1 (HR = 1.25 95% CI: 1.01-1.54, p = 0.041) were significant predictors of subsequent malignancy. The cumulated incidence at 5 years landmark time was 17% for those subjects with elevated CEA, CA19-9 and TIMP-1 versus 6.7% for those with low levels of all. When the model was applied to the validation set the cumulated 5-year incidence was 10.5% for subjects with elevated CEA, CA19-9 and TIMP-1 and 5.6% for subjects with low levels of all biomarkers. Further analysis demonstrated a significant interaction between TIMP-1 and age in the training set. The age dependency of TIMP-1 indicated a greater risk of malignancy in younger subjects if the biomarker was elevated. This observation was validated in the second set. CONCLUSION: Elevated cancer-associated protein biomarker levels in subjects with non-malignant findings at large bowel endoscopy identifies subjects at increased risk of being diagnosed with subsequent primary malignancy. CEA, CA19-9 and TIMP-1 were significant predictors of malignant disease in this analysis. TIMP-1 was found dependent on age. The results were validated in an independent symptomatic population.

Investigations on the in vivo metabolism of 5α-androst-2-en-17-one.

RATIONALE: The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2ß,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. METHODS: The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. RESULTS: More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5ß-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5ß-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. CONCLUSIONS: The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5ß-configuration has not been reported so far and should be further investigated.

Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes.

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.

Carbon isotope ratios of endogenous steroids found in human serum-method development, validation, and reference population-derived thresholds.

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)-based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.

Current Insights into the Steroidal Module of the Athlete Biological Passport.

For decades, the class of anabolic androgenic steroids has represented the most frequently detected doping agents in athletes' urine samples. Roughly 50% of all adverse analytical findings per year can be attributed to anabolic androgenic steroids, of which about 2/3 are synthetic exogenous steroids, where a qualitative analytical approach is sufficient for routine doping controls. For the remaining 1/3 of findings, caused by endogenous steroid-derived analytical test results, a more sophisticated quantitative approach is required, as their sheer presence in urine cannot be directly linked to an illicit administration. Here, the determination of urinary concentrations and concentration ratios proved to be a suitable tool to identify abnormal steroid profiles. Due to the large inter-individual variability of both concentrations and ratios, population-based thresholds demonstrated to be of limited practicability, leading to the introduction of the steroidal module of the Athlete Biological Passport. The passport enabled the generation of athlete-specific individual reference ranges for steroid profile parameters. Besides an increase in sensitivity, several other aspects like sample substitution or numerous confounding factors affecting the steroid profile are addressed by the Athlete Biological Passport-based approach. This narrative review provides a comprehensive overview on current prospects, supporting professionals in sports drug testing and steroid physiology.

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis.

RATIONALE: The misuse of 7-oxo-DHEA (3ß-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7ß-OH-DHEA were identified, necessitating further investigations. METHODS: As both 7-oxo-DHEA and 7ß-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. RESULTS: Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7ß-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5α-androstane-3ß,7ß-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7ß-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7ß-OH-DHEA. CONCLUSIONS: As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7ß-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens.

Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016.

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Increased serological, cancer-associated protein biomarker levels at diagnosis of large bowel adenoma: Risk of subsequent primary malignancy?

BACKGROUND: Blood-based, cancer-associated biomarkers may detect subjects at risk of having neoplastic diseases. The aim of the present study was to evaluate whether elevated serological protein biomarker levels may identify adenoma patients, who are at increased risk of being diagnosed with subsequent primary malignancy. METHODS: Levels of CEA, CA19-9, TIMP-1 and YKL-40 were determined in blood samples collected prior to diagnostic bowel endoscopy due to symptoms of colorectal neoplasia. Follow-up time was ten years, and identified adenoma patients, who were diagnosed with subsequent primary intra- or extra-colonic malignant diseases. The biomarker levels were also determined in 400 subjects, who underwent diagnostic colonoscopy, had clean colorectum and were without apparent co-morbidity; these levels were used as reference levels. In the present study, biomarkers were interpreted as elevated when levels were above the reference intervals adjusting for age and gender. The 1-year and 5-years cumulative incidences were calculated. RESULTS: Primary malignancies were identified in 175 (19%) of the 923 subjects diagnosed with adenomas at the primary bowel endoscopy. In detail, 20 of the 175 subjects were diagnosed with colorectal cancer (CRC) and 155 subjects with extra-colonic cancers. Thirty patients were diagnosed with malignancy within the first year. Three groups were established: 0: no elevated biomarkers; 1: 1 of the 4 biomarkers elevated; and 2: ≥2 biomarkers elevated. The cumulative 5-years incidence of malignancy was: 0: 6.9%; 1: 11.8%; and 2: 17.5% (p = .0009). CONCLUSION: Elevated blood-based, cancer-associated protein biomarker levels in subjects diagnosed with adenomas at large bowel endoscopy identifies subjects at increased risk of being diagnosed with subsequent primary malignancy.

Analysis of endogenous steroids in urine by means of multi-immunoaffinity chromatography and isotope ratio mass spectrometry for sports drug testing.

Detecting the administration of naturally occurring but synthetically derived steroids (e.g., testosterone) in routine doping controls is particularly laborious and time-consuming. Carbon isotope signatures determined by isotope ratio mass spectrometry (IRMS) have been established as the method of choice to generate confirmatory evidence in case of suspicious or atypical findings in steroid profile analyses; however, IRMS measurements require sophisticated sample preparation methods employing up to two high-performance liquid chromatography (HPLC) purification steps. Here, an alternative sample preparation approach is presented. Immunoaffinity chromatography (IAC) was employed to reduce the batch analysis time by omitting the time-consuming HPLC purification steps, while pre- and post-IAC sample handling followed published protocols. IAC exploits specific antibody-immunogen interactions, and the option of combining three immunoaffinity gels containing specific antibodies for testosterone, pregnanediol, and 11-ketoetiocholanolone into a multi-immunoaffinity sample preparation approach was assessed. Due to cross reactivities, also etiocholanolone, androsterone, 5ß-androstanediol, and 5α-androstanediol were co-extracted and included in the testing protocol. The method was validated by determining precision, recovery, and carry over, and performing linear mixing models. IAC was found to be applicable to the determination of carbon isotope ratios in doping controls and the approach allowed for an accelerated sample preparation. Graphical abstract.

Carbon isotope ratios of endogenous steroids in Belgian Blue and Holstein cattle: Method development, reference population studies and application to steroid misuse control.

RATIONALE: The misuse of growth promoters in livestock and breeding animals is prohibited according to the laws of the European Union. Among these growth promoters, the detection of endogenous steroids like testosterone, estradiol or progesterone remains especially challenging as concentration-based urinary thresholds may not provide conclusive results due to large inter-individual variations. In addition to the detection of intact steroid esters in blood or hair, carbon isotope ratio (CIR) determination of urinary steroids has commonly been the method of choice. METHODS: A comprehensive sample clean-up procedure was developed and validated, which enables for the first time simultaneous CIR measurements of testosterone metabolites (17α-hydroxyandrost-4-en-3-one, 3α-hydroxy-5ß-androstan-17-one and 5α-androstane-3ß,17α-diol), the estradiol metabolite 17α-estradiol (ESTR) and the progesterone metabolite 5ß-pregnane-3α,20α-diol (PD) from a single urine specimen. As endogenous reference compounds 3ß-hydroxyandrost-5-en-17-one and 5-androstene-3ß,17α-diol (5EN) were chosen. The method was validated by means of linear mixing models and a reference population encompassing n = 53 Belgian Blue and Holstein cattle was investigated to enable the calculation of population-based Δ13 C thresholds. RESULTS: The combined measurement uncertainty determined for the Δ13 C-values of all steroids under investigation was found to be <0.8 ‰. Within the reference population studies, 5EN was demonstrated to be the most promising endogenous reference compound resulting in comparably low Δ-values and accompanying thresholds. For PD, a surprisingly high number of samples (n = 9) yielded significantly 13 C-depleted values and ESTR was only detectable in n = 13 samples. Proof-of-concept was accomplished by investigating two post-administration samples. CONCLUSIONS: This first comprehensive investigation on the CIRs of endogenous urinary steroids demonstrated once more the potential of isotope ratios in aiding discrimination between endogenously produced and exogenously administered steroids. By means of the reference population-derived CIRs, it is possible to apply cattle-specific thresholds to differentiate between treated and non-treated animals.

Mass spectrometric characterization of the selective androgen receptor modulator (SARM) YK-11 for doping control purposes.

RATIONALE: Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics targeting inter alia conditions referred to as cachexia and sarcopenia. Due to their anabolic properties, the use of SARMs is prohibited in sports as regulated by the World Anti-Doping Agency (WADA), and doping control laboratories test for these anabolic agents in blood and urine. In order to accomplish and maintain comprehensive test methods, the characterization of new drug candidates is critical for efficient sports drug testing. Hence, in the present study the mass spectrometric properties of the SARM YK-11 were investigated. METHODS: YK-11 was synthesized according to literature data and three different stable-isotope-labeled analogs were prepared to support the mass spectrometric studies. Using high-resolution/high-accuracy mass spectrometry following electrospray ionization as well as electron ionization, the dissociation pathways of YK-11 were investigated, and characteristic features of its (product ion) mass spectra were elucidated. These studies were flanked by density functional theory (DFT) computation providing information on proton affinities of selected functional groups of the analyte. RESULTS AND CONCLUSIONS: The steroidal SARM YK-11 was found to readily protonate under ESI conditions followed by substantial in-source dissociation processes eliminating methanol, acetic acid methyl ester, and/or ketene. DFT computation yielded energetically favored structures of the protonated species resulting from the aforementioned elimination processes particularly following protonation of the steroidal D-ring substituent. Underlying dissociation pathways were suggested, supported by stable-isotope labeling of the analyte, and diagnostic product ions for the steroidal nucleus and the D-ring substituent were identified. Further, trimethylsilylated YK-11 and its deuterated analogs were subjected to electron ionization high-resolution/high-accuracy mass spectrometry, complementing the dataset characterizing this new SARM. The obtained fragment ions resulted primarily from A/B- and C/D-ring structures of the steroidal nucleus, thus supporting future studies e.g. concerning metabolic pathways of the substance. Copyright © 2017 John Wiley & Sons, Ltd.

Increased serological cancer-associated biomarker levels at large bowel endoscopy and risk of subsequent primary cancer (†).

BACKGROUND: Frequently, subjects offered colonoscopy due to symptoms of colorectal neoplasia are diagnosed with diverticula. The symptoms may, however, also be related to extra-colonic neoplasia. The present retrospective study evaluated a possible association between increased levels of predefined biomarkers in subjects diagnosed with diverticula and risk of developing a primary malignant disease. METHODS: During 2004/2005, about 4509 subjects were included in a multicenter study with collection of blood samples before bowel endoscopy. The aim was to evaluate a relation between the protein biomarkers CEA, TIMP-1, CA19-9 and YKL-40 and findings at endoscopy. Diverticula were diagnosed in 1021 subjects. By 31 December 2012, subjects who had developed primary malignancy were identified retrospectively and relation between biomarker levels at endoscopy and risk of developing primary malignancy was calculated. The relation with the four biomarkers was divided into three groups: 0 = none increased; 1 = one increased and 2 = two or more increased. RESULTS: In the observation period, 148 subjects developed a primary malignant disease. Univariable analyzes of the biomarker levels showed that CEA, TIMP-1 and CA19-9 were significantly associated with development of primary malignancy. A multivariable analysis showed that increased levels were associated with development of malignancy (p < 0.0001). The 1- and 5-year cumulative risks of being diagnosed with a primary malignancy were: group 0: 1.1%/5.5%; group 1: 4.2%/10.1% and group 2: 11.4%/18.8%, respectively. CONCLUSION: Increased levels of CEA, TIMP-1 and CA19-9 at endoscopy with findings of diverticula were associated with a significantly increased risk of being diagnosed with a subsequent primary malignant disease.

Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry.

Since its release as anti-anemic drug, recombinant erythropoietin (rEPO) gradually entered the illicit way to sports competitions as endurance-enhancing drug. Novel modifications biopharmaceutically introduced into the rEPO molecule in the form of carbohydrate or polyethylene glycol moieties made robust and sensitive test methods vital to doping controls in order to provide the necessary tools enabling the conviction of dishonest athletes. Modern protein analysis by means of gel electrophoretic separation and western blotting represents the status quo in rEPO anti-doping analysis. However, new therapeutically promising erythropoietin receptor activating compounds have been developed that exhibit cytokine hormone-mimicking properties but lack any protein structure. Progression to evade parenteral application and substitute for rEPO by low molecular mass and orally available compounds is still one of the major objectives in pharmaceutical research. In this approach, four promising in-house synthesized nonpeptidic erythropoietin mimetic agents, namely compound 129, compound 163, A1B10C1, and A5B10C4 were thoroughly evaluated by employing high-resolution/high-accuracy liquid chromatography tandem mass spectrometry experiments. Characteristic product ions were determined supporting the identification of these drugs and putative metabolites as well as related compounds in future doping controls. Test methods employing direct urine injection and receptor affinity purification strategies were assessed, which demonstrated that EPO receptor purification is of limited utility for nonpeptidic EPOR agonists while direct urine injection allowed for comprehensive method characterization. Thereby, achieved limits of detection were 1 ng/mL for compounds 129/163 and 5 ng/mL for A1B10C1/A5B10C4.

Urine analysis concerning xenon for doping control purposes.

RATIONALE: On September 1(st) 2014, a modified Prohibited List as established by the World Anti-Doping Agency (WADA) became effective featuring xenon as a banned substance categorized as hypoxia-inducible factor (HIF) activator. Consequently, the analysis of xenon from commonly provided doping control specimens such as blood and urine is desirable, and first data on the determination of xenon from urine in the context of human sports drug testing, are presented. METHODS: In accordance to earlier studies utilizing plasma as doping control matrix, urine was enriched to saturation with xenon, sequentially diluted, and the target analyte was detected as supported by the internal standard d6 -cyclohexanone by means of gas chromatography/triple quadrupole mass spectrometry (GC/MS/MS) using headspace injection. Three major xenon isotopes at m/z 128.9, 130.9 and 131.9 were targeted in (pseudo) selected reaction monitoring mode enabling the unambiguous identification of the prohibited substance. Assay characteristics including limit of detection (LOD), intraday/interday precision, and specificity as well as analyte recovery under different storage conditions were determined. Proof-of-concept data were generated by applying the established method to urine samples collected from five patients before, during and after (up to 48 h) xenon-based general anesthesia. RESULTS: Xenon was traceable in enriched human urine samples down to the detection limit of approximately 0.5 nmol/mL. The intraday and interday imprecision values of the method were found below 25%, and specificity was demonstrated by analyzing 20 different blank urine samples that corroborated the fitness-for-purpose of the analytical approach to unequivocally detect xenon at non-physiological concentrations in human urine. The patients' urine specimens returned 'xenon-positive' test results up to 40 h post-anesthesia, indicating the limits of the expected doping control detection window. CONCLUSIONS: Since xenon has been considered a prohibited substance according to WADA regulations in September 2014, its analysis from common specimens of routine sports drug testing is desirable. In previous studies, its traceability in whole blood and plasma was shown, and herein a complementary approach utilizing doping control urine samples for the GC/MS/MS analysis of xenon was reported.

Carbon isotope ratio analysis of steroids by high-temperature liquid chromatography-isotope ratio mass spectrometry.

Generally, compound-specific isotope analysis of steroids is carried out by gas chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the steroids prior to the measurement is compulsory, and a correction of the isotopic data is often necessary. To overcome this limitation, we present a new approach of high-temperature liquid chromatography coupled with photodiode array detection and isotope ratio mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone, androsterone, and 5ß-pregnane-3α,17α,20α-triol was fully separated on a C4 column under high-temperature elution with water as the sole eluent. The accuracy for isotope analysis (±0.5 ‰) was around 20 µg g(-1) for testosterone, epitestosterone (79 ng steroid absolute on column), and 30 µg g(-1) for 19-norandrosterone, androsterone, and 5ß-pregnane-3α,17α,20α-triol (119 ng steroid absolute on column). The applicability of the method was tested by measuring a pharmaceutical gel containing testosterone. With this work, the scope of LC/IRMS applications has been extended to nonpolar compounds.

Measuring xenon in human plasma and blood by gas chromatography/mass spectrometry.

RATIONALE: Due to the favorable pharmacokinetic properties and minimal side effects of xenon, its use in modern anesthesia has been well accepted, and recent studies further demonstrated the intra- and postoperative neuro-, cardio-, and reno-protective action of the noble gas. Since the production of the hypoxia-inducible factor 1α (HIF-1α) and its downstream effector erythropoietin as well as noradrenalin reuptake inhibition have been found to play key roles in this context, the question arose as to whether the use of xenon is a matter for doping controls and preventive doping research. The aim of the present study was hence to evaluate whether the (ab)use of xenon can be detected from doping control samples with the instrumentation commonly available in sports drug testing laboratories. METHODS: Plasma was saturated with xenon according to reported protocols, and the target analyte was measured by means of gas chromatography/time-of-flight and triple quadrupole mass spectrometry with headspace injection. Recording the accurate mass of three major xenon isotopes at m/z 128.9048, 130.9045 and 131.9042 allowed for the unequivocal identification of the analyte and the detection assay was characterized concerning limit of detection (LOD), intraday precision, and specificity as well as analyte recovery under different storage conditions. RESULTS: Xenon was detected in fortified plasma samples with detection limits of approximately 0.5 nmol/mL to 50 nmol/mL, depending on the type of mass spectrometer used. The method characteristics of intraday precision (coefficient of variation <20%) and specificity demonstrated the fitness-for-purpose of the analytical approach to unambiguously detect xenon at non-physiological concentrations in human plasma and blood. Eventually, authentic plasma and blood samples collected pre-, intra-, and post-operative (4, 8, and 24 h) were positively analyzed after storage for up to 30 h, and provided proof-of-concept for the developed assay. CONCLUSIONS: If relevant to doping controls, xenon can be determined from plasma and blood samples, i.e. common specimens of routine sports drug testing in the context of Athlete Biological Passport (ABP) analyses. Optimization of sampling and analytical procedures will allow the detection limit to be further improved and potentially enable accurate quantification of the anesthetic agent.

Determination of ¹³C/¹² C ratios of endogenous urinary 5-amino-imidazole-4-carboxamide 1ß-D-ribofuranoside (AICAR).

RATIONALE: AICAR (5-aminoimidazole-4-carboxamide 1ß-D-ribofuranoside) is prohibited in sport according to rules established by the World Anti-Doping Agency. Doping control laboratories identify samples where AICAR abuse is suspected by measuring its urinary concentration and comparing the observed level with naturally occurring concentrations. As the inter-individual variance of urinary AICAR concentrations is large, this approach requires a complementary method to unambiguously prove the exogenous origin of AICAR. Therefore, a method for the determination of carbon isotope ratios (CIRs) of urinary AICAR has been developed and validated. METHODS: Concentrated urine samples were fractionated by means of liquid chromatography for analyte cleanup. Derivatization of AICAR yielding the trimethylsilylated analog was necessary to enable CIR determinations by gas chromatography/combustion/isotope ratio mass spectrometry. The method was tested for its repeatability and stability over time and a linear mixing model was applied to test for possible isotopic discrimination. A reference population of n = 63 males and females was investigated to calculate appropriate reference limits to differentiate endogenous from exogenous urinary AICAR. These limits were tested by an AICAR elimination study. RESULTS: The developed method fulfills all the requirements for adequate sports drug testing and was found to be fit for purpose. The investigated reference population showed a larger variability in the CIR of AICAR than of the endogenous steroids. Nevertheless, the calculated thresholds for differences between AICAR and endogenous steroids can be applied straightforwardly to evaluate suspicious doping control samples with the same statistical confidence as established e.g. for testosterone misuse. These thresholds enabled the detection of a single oral AICAR administration for more than 40 h. CONCLUSIONS: Determination of thee CIRs is the method of choice to distinguish between an endogenous and an exogenous source of urinary AICAR. The developed method will enable investigations into doping control samples with elevated urinary concentrations of AICAR and clearly differentiate between naturally produced/elevated and illicitly administered AICAR.

Identification of black market products and potential doping agents in Germany 2010-2013.

PURPOSE: The desire to increase the athletic performance, to 'optimize' an individual's appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities. METHODS: From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). RESULTS: Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin ß4, and a fusion protein of unknown biological activity. CONCLUSIONS: Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the 'customer'.

Liquid chromatography-high resolution/ high accuracy (tandem) mass spectrometry-based identification of in vivo generated metabolites of the selective androgen receptor modulator ACP-105 for doping control purposes.

Selective androgen receptor modulators (SARMs) represent an emerging class of therapeutics which have been prohibited in sport as anabolic agents according to the regulations of the World Anti-Doping Agency (WADA) since 2008. Within the past three years, numerous adverse analytical findings with SARMs in routine doping control samples have been reported despite missing clinical approval of these substances. Hence, preventive doping research concerning the metabolism and elimination of new therapeutic entities of the class of SARMs are vital for efficient and timely sports drug testing programs as banned compounds are most efficiently screened when viable targets (for example, characteristic metabolites) are identified. In the present study, the metabolism of ACP-105, a novel SARM drug candidate, was studied in vivo in rats. Following oral administration, urine samples were collected over a period of seven days and analyzed for metabolic products by Liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Samples were subjected to enzymatic hydrolysis prior to liquid-liquid extraction and a total of seven major phase-I metabolites were detected, three of which were attributed to monohydroxylated and four to bishydroxylated ACP-105. The hydroxylation sites were assigned by means of diagnostic product ions and respective dissociation pathways of the analytes following positive or negative ionization and collisional activation as well as selective chemical derivatization. The identified metabolites were used as target compounds to investigate their traceability in a rat elimination urine samples study and monohydroxylated and bishydroxylated species were detectable for up to four and six days post-administration, respectively.

Employing 11-Ketotestosterone as a Target Analyte for Adrenosterone (11OXO) Administration in Doping Controls.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11ß-hydroxy-androsterone and 11ß-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.