Delayed Drowsiness After Normobaric Hypoxia Training in an F/A-18 Hornet Simulator.

BACKGROUND: In military aviation, due to high-altitude flight operations, hypoxia training is mandatory and nowadays is mainly done as normobaric hypoxia training in flight simulators. During the last decade, scientific data has been published about delayed recovery after normobaric hypoxia, known as a "hypoxia hangover." Sopite syndrome is a symptom complex that develops as a result of exposure to real or apparent motion, and it is characterized by yawning, excessive drowsiness, lassitude, lethargy, mild depression, and a reduced ability to focus on an assigned task.CASE REPORT: In this study, we present the case of a 49-yr-old pilot who participated in normobaric hypoxia refreshment training in an F/A-18C Hornet simulator and experienced delayed drowsiness, even 3 h after the training.DISCUSSION: This case report demonstrates the danger of deep hypoxia. Hypoxia training instructions should include restrictions related to driving a car immediately after hypoxia training. In addition, hypoxia may lower the brain threshold for sopite syndrome.Varis N, Leinonen A, Perälä J, Leino TK, Husa L, Sovelius R. Delayed drowsiness after normobaric hypoxia training in an F/A-18 Hornet simulator. Aerosp Med Hum Perform. 2023; 94(9):715-718.

A New Method for Combined Hyperventilation and Hypoxia Training in a Tactical Fighter Simulator.

INTRODUCTION: Physiological episodes are an issue in military aviation. Some non-pressure-related in-flight symptoms are proved to be due to hyperventilation rather than hypoxia. The aim of this study was to validate a new training method provoking hyperventilation during normobaric hypoxia (NH) training in an F/A-18 Hornet simulator.METHODS: In a double-blind setting, 26 fighter pilots from the Finnish Air Force performed 2 setups in a WTSAT simulator in randomized order with full flight gear. Without the pilot's knowledge, 6% O2 in nitrogen or 6% O2 + 4% CO2 in nitrogen was turned on. Ventilation (VE) was measured before, during, and after hypoxia. Spo2 and ECG were monitored and symptoms documented. The subjects performed a tactical identification flight until they recognized symptoms of hypoxia. Thereafter, they performed hypoxia emergency procedures with 100% O2 and returned to the base with a GPS malfunction and executed an instrument landing system (ILS) approach with the waterline HUD mode evaluated by the flight instructor on a scale of 1 to 5.RESULTS: Ventilation increased during normobaric hypoxia (NH) from 12 L · min-1 to 19 L · min-1 at Spo2 75% with 6% O2, and from 12 L · min-1 to 26 L · min-1 at Spo2 77% with 6% O2 + 4% CO2. ILS flight performance was similar 10 min after combined hyperventilation and hypoxia (3.1 with 6% O2 + 4% CO2 and 3.2 with 6% O2). No adverse effects were reported during the 24-h follow-up.DISCUSSION: Hyperventilation-provoking normobaric hypoxia training is a new and well-tolerated method to meet NATO Standardization Agreement hypoxia training requirements.Leinonen AM, Varis NO, Kokki HJ, Leino TK. A new method for combined hyperventilation and hypoxia training in a tactical fighter simulator. Aerosp Med Hum Perform. 2022; 93(9):681-687.

Hyperventilation and Hypoxia Hangover During Normobaric Hypoxia Training in Hawk Simulator.

Introduction: In military aviation during high-altitude operations, an oxygen or cabin pressure emergency can impair brain function and performance. There are variations in individuals' physiological responses to low partial pressure of oxygen and hypoxia symptoms can vary from one exposure to another. The aim of this study was to evaluate how normobaric hypoxia (NH) affects pilots' minute ventilation and 10 min afterwards on Instrument Landing System (ILS) flight performance in Hawk simulator during a tactical flight sortie. Methods: Fifteen volunteer fighter pilots from the Finnish Air Force participated in this double blinded, placebo controlled and randomized study. The subjects performed three flights in a tactical Hawk simulator in a randomized order with full flight gear, regulators and masks on. In the middle of the flight without the subjects' knowledge, 21% (control), 8% or 6% oxygen in nitrogen was turned on. Minute ventilation (VE) was measured before, during NH and after NH. Forehead peripheral oxygen saturation (SpO2), wireless ECG and subjective symptoms were documented. The flights were conducted so that both subjects and flight instructors were blinded to the gas mixture. The pilots performed tactical maneuvers at simulated altitude of 20,000 ft or 26,000 ft until they recognized the symptoms of hypoxia. Thereafter they performed hypoxia emergency procedures with 100% oxygen and returned to base (RTB). During the ILS approach, flight performance was evaluated. Results: The mean VE increased during NH from 12.9 L/min (21% O2 on the control flight) to 17.8 L/min with 8% oxygen (p < 0.01), and to 21.0 L/min with 6% oxygen (p < 0.01). Ten minutes after combined hyperventilation and hypoxia, the ILS flight performance decreased from 4.4 (control flight) to 4.0 with 8% oxygen (p = 0.16) and to 3.2 with 6% oxygen (p < 0.01). A significant correlation (r = -0.472) was found between the subjects' VE during 6% oxygen exposure and the ILS flight performance. Discussion: Hyperventilation during NH has a long-lasting and dose-dependent effect on the pilot's ILS flight performance, even though the hypoxia emergency procedures are executed 10 min earlier. Hyperventilation leads to body loss of carbon dioxide and hypocapnia which may even worsen the hypoxia hangover.

Normobaric hypoxia training in military aviation and subsequent hypoxia symptom recognition.

Altitude hypoxia episodes are increasingly common in military aviation. Hypoxia training is mandatory for fighter pilots, but evidence-based data on the effects of training are scarce. The purpose of this study was to validate the normobaric hypoxia (NH) training effect. Data were collected from 89 pilots from the Finnish Air Force (FINAF). This survey was conducted in a tactical F/A-18C Hornet simulator in two sessions under normobaric conditions, in which the pilots performed flight missions and breathed 21% oxygen (O2) in nitrogen (N2), and blinded to the pilot, the breathing gas was changed to a hypoxic mixture containing either 8, 7 or 6% O2 in N2. The time taken to notice hypoxia symptoms and peripheral capillary O2 saturation was measured. A mean of 2.4 years after the initial training, pilots recognised their hypoxic symptoms 18 s quicker with 8% O2 mixture, 20 s quicker with 7% O2 and 10 s quicker with 6% O2. Our data indicate that NH training in a flight simulator helps pilots to recognise hypoxia symptoms earlier, and may, thus, enhance flight safety.Practitioner Summary: We show that hypoxia training enhances pilots' ability to recognise symptoms of acute normobaric hypoxic exposure up to 2.4 years after an initial NH training session. Based on these data, refreshment NH training is nowadays mandatory every 3 years in the FINAF as opposed to the North Atlantic Treaty Organisation (NATO) Standardisation Agreement (STANAG) requirement of 5-year intervals between hypoxia trainings.Abbreviations: O2: oxygen; TUC; time of usefull consciousness; SpO2: peripheral capillary oxygen saturation; NATO: North Atlantic Treaty Organization; STANAG: stanrdization agreement; HH: hypobaric hypoxia; NH: normobaric hypoxia; FINAF: finnish air force; N2: nitrogen; ECG: electrocardiogram; CI: confidence interval; SD: standard deviation.

Analysis of luteinizing hormone (LH): Validation of a commercial ELISA kit for LH analysis and quantification in doping control samples.

Luteinizing hormone (LH) is a dimeric glycoprotein produced and secreted by the pituitary gland, with a molecular weight of approximately 30 000 Da. The main clinical use for exogenous LH-administration is typically linked to the treatment of infertility, in both men and women. The desired effect of LH misuse in sport is due to the enhancement of testicular production of testosterone. Elevated LH levels may also indicate the usage of gonadotropin-releasing factors or estrogen blockers. Therefore, LH is listed by the World Anti-Doping Agency (WADA) as a prohibited substance in male athletes, and according to the WADA technical document, laboratories should determine the the total LH concentrations in urine. The TD lists two different assays that are suitable for measuring total LH in urine, Delfia and Siemens Immulite. Other fit-for-purpose assays can be used, as long as they are capable of detecting total LH in urine. In case an assay not listed in the TD will be used, population-based reference values have to be determined in the validation procedure. In this study a new immunoassay was validated for the measurement of LH in urine. The assay (AccuBind ELISA Microwells, Luteinizing Hormone, Monobind Inc.), originally intended for serum, showed adequate sensitivity and was proven fit-for-purpose in routine doping control. Population-based distribution of the assay was in good agreement with the results of Delfia and Immulite assays, for which the method-specific cut-off-values are 40 IU/L (Delfia) and 60 IU/L (Siemens Immulite).

What Data to Use for Forest Conservation Planning? A Comparison of Coarse Open and Detailed Proprietary Forest Inventory Data in Finland.

The boreal region is facing intensifying resource extraction pressure, but the lack of comprehensive biodiversity data makes operative forest conservation planning difficult. Many countries have implemented forest inventory schemes and are making extensive and up-to-date forest databases increasingly available. Some of the more detailed inventory databases, however, remain proprietary and unavailable for conservation planning. Here, we investigate how well different open and proprietary forest inventory data sets suit the purpose of conservation prioritization in Finland. We also explore how much priorities are affected by using the less accurate but open data. First, we construct a set of indices for forest conservation value based on quantitative information commonly found in forest inventories. These include the maturity of the trees, tree species composition, and site fertility. Secondly, using these data and accounting for connectivity between forest types, we investigate the patterns in conservation priority. For prioritization, we use Zonation, a method and software for spatial conservation prioritization. We then validate the prioritizations by comparing them to known areas of high conservation value. We show that the overall priority patterns are relatively consistent across different data sources and analysis options. However, the coarse data cannot be used to accurately identify the high-priority areas as it misses much of the fine-scale variation in forest structures. We conclude that, while inventory data collected for forestry purposes may be useful for forest conservation purposes, it needs to be detailed enough to be able to account for more fine-scaled features of high conservation value. These results underline the importance of making detailed inventory data publicly available. Finally, we discuss how the prioritization methodology we used could be integrated into operative forest management, especially in countries in the boreal zone.

Targeting misuse of 2-amino-N-ethyl-1-phenylbutane in urine samples: in vitro-in vivo correlation of metabolic profiles and development of LC-TOF-MS method.

A phenyethylamine derivative, 2-amino-N-ethyl-1-phenylbutane (2-AEPB), has recently been detected in doping control and drugs-of-abuse samples, and identified as a non-labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2-AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2-AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N-oxidation was also observed (M3). In vitro-in vivo correlation was studied in an excretion study with a single, oral dose of 2-AEPB-containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2-AEPB and M1 were mainly found in free urinary fraction, whereas higher inter-individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide- and sulpho-conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2-AEPB.

Analysis of anabolic steroids in urine by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry with chlorobenzene as dopant.

A gas chromatography-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-µAPPI-MS/MS) method was developed for the analysis of anabolic androgenic steroids in urine as their trimethylsilyl derivatives. The method utilizes a heated nebulizer microchip in atmospheric pressure photoionization mode (µAPPI) with chlorobenzene as dopant, which provides high ionization efficiency by producing abundant radical cations with minimal fragmentation. The performance of GC-µAPPI-MS/MS was evaluated with respect to repeatability, linearity, linear range, and limit of detection (LOD). The results confirmed the potential of the method for doping control analysis of anabolic steroids. Repeatability (RSD<10%), linearity (R(2)≥0.996) and sensitivity (LODs 0.05-0.1ng/mL) were acceptable. Quantitative performance of the method was tested and compared with that of conventional GC-electron ionization-MS, and the results were in good agreement.

Analysis of human chorionic gonadotropin (hCG): application of routine immunological methods for initial testing and confirmation analysis in doping control.

Human chorionic gonadotropin (hCG) is dimeric glycoprotein produced by placenta in pregnancy and also in low levels by pituitary gland. The main clinical use for exogenous hCG-administration is typically linked to infertility. The desired effect of hCG misuse in sport is due to the enhancement of testicular production of testosterone. Therefore, hCG is listed by the World Anti-Doping Agency (WADA) as a prohibited substance in male athletes and according to the recently published WADA guideline urinary concentrations of hCG > 5 IU/L may be an indicator of doping. In this study two independent immunoassays were used to implement the new WADA guideline. The assay for initial testing (Siemens Immulite 2000 XPi hCG assay) recognizes various hCG variants (e.g. hCG and ß-core fragment of hCG) whereas the confirmatory assay (PerkinElmer DELFIA Xpress hCG) is sensitive to intact and nicked hCG only. Both assays showed adequate sensitivity and were proven fit-for-purpose in routine doping control. Population-based distribution of the assays was in good agreement with results of earlier studies and supported well the current threshold of 5 IU/L.

Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography-in-source collision-induced dissociation-time-of-flight mass spectrometry.

The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic-mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R(2) > 0.99 for both analytes. Limits of detection were 100 and 250 µg mL(-1) for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC-ISCID-TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile.

Feasibility of gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry in analysis of anabolic steroids.

Mass spectrometers equipped with atmospheric pressure ion sources (API-MS) have been designed to be interfaced with liquid chromatographs (LC) and have rarely been connected to gas chromatographs (GC). Recently, we introduced a heated nebulizer microchip and showed its potential to interface liquid microseparation techniques and GC with API-MS. This study demonstrates the feasibility of GC-microchip atmospheric pressure photoionization-tandem mass spectrometry (GC-µAPPI-MS/MS) in the analysis of underivatized anabolic steroids in urine. The APPI microchip provides high ionization efficiency and produces abundant protonated molecules or molecular ions with minimal fragmentation. The feasibility of GC-µAPPI-MS/MS in the analysis of six selected anabolic steroids in urine samples was studied with respect to intra-batch repeatability, linearity, linear range, and limit of detection (LOD). The method showed good sensitivity (LODs 0.2-1 ng/mL), repeatability (relative standard deviation<10%), and linearity (regression coefficient≥0.9995) and, therefore, high potential for the analysis of anabolic steroids. Quantitative performance of the method was tested with two authentic urine samples, and the results were in good agreement with those obtained with conventional GC-electron ionization-MS after derivatization.

Characterization of microbial contaminants in urine.

Modern, molecular microbiological methods were applied to urine samples from control subjects and athletes for characterization of the microbial community. High abundance of lactobacilli, enterococci, and enterobacteria was detected in urine samples, suggesting that gastrointestinal and urogenital tracts act as contamination sources. Athlete samples, but not control samples, showed an abundance of pseudomonads, a bacterial group reported to metabolize steroids. Overall, the bacteria detected are known to be capable of altering steroid profiles, emphasizing the importance of good hygiene at sampling in reliable doping control.

UDP-glucuronosyltransferases in conjugation of 5alpha- and 5beta-androstane steroids.

We have examined the glucuronidation of androsterone (5alpha-androstane-3alpha-ol-17-one), etiocholanolone (5beta-androstane-3alpha-ol-17-one), 5alpha-androstane-3alpha-,17beta-diol (5alpha-diol), and 5beta-androstane-3alpha-, 17beta-diol (5beta-diol) by 19 recombinant human UDP-glucuronosyltransferases (UGTs). The results reveal large differences in stereo- and regioselectivity between UGT2B7, UGT2B15, and UGT2B17. UGT2B7 conjugated all four androgens at the 3-OH but not at the 17-OH that is available in both diols. UGT2B7 exhibited a higher glucuronidation rate toward the steroids with a flat backbone, androsterone and 5alpha-diol, compared with etiocholanolone and 5beta-diol, which have a bent backbone. UGT2B17 readily glucuronidated androsterone and, particularly, etiocholanolone at the 3-OH, but in the two diols it exhibited high preference for the 17-OH and low glucuronidation rate at the 3-OH. UGT2B15 did not glucuronidate any of the studied four androgens at the 3-OH, but it did conjugate both diols at the 17-OH, with a clear preference for 5alpha-diol. Of the UGT1A subfamily, only UGT1A4 catalyzed the glucuronidation of androsterone and 5alpha-diol at measurable rates, even if low. UGT2A1 and UGT2A2 glucuronidated most compounds in this study, but mostly at rather low rates. An exception was the glucuronidation of etiocholanolone by UGT2A1 that revealed a very low substrate affinity in combination with very high V(max) value. The results shed new light on the substrate selectivity of individual UGTs in steroid glucuronidation. In addition they bear implications for doping analyses and its dependence of genetic polymorphism because testosterone is a precursor in the biosynthesis of these four androgens, whereas the contribution of UGT2B17 to their glucuronidation varies greatly.

Synthesis and characterization of hydroxylated mesocarb metabolites for doping control.

The synthesis and method of analysis of hydroxylated mesocarb metabolites are described. Six potential hydroxylated mesocarb metabolites were prepared, characterized, and compared with the mesocarb metabolites synthesized enzymatically in vitro using human liver proteins and also compared with metabolites extracted from human urine after oral administration of mesocarb. p-Hydroxymesocarb was the most prevalent metabolite (conjugated and non-conjugated) observed. With respect to doping analysis, synthesis of p-hydroxymesocarb, the main urinary metabolite of mesocarb, and its availability as a reference material is important.

UDP-glucuronosyltransferases (UGTs) 2B7 and UGT2B17 display converse specificity in testosterone and epitestosterone glucuronidation, whereas UGT2A1 conjugates both androgens similarly.

Testosterone and epitestosterone are endogenous steroids that differ in the configuration of the hydroxyl-bearing carbon at C-17. Testosterone is the predominant male sex hormone, whereas the role of epitestosterone is largely unclear. In humans, both androgens are excreted mainly as glucuronide conjugates and the urinary ratio of testosterone to epitestosterone (T/E), used to expose illicit testosterone abuse by male athletes, indicates the relative concentrations of the respective glucuronides. Some male athletes have T/E values greater than the accepted threshold value (4.0), even without testosterone abuse. We have analyzed athletes' urine samples and found that the main reason for such false-positive results in doping tests was a low epitestosterone glucuronide concentration not a high level of testosterone glucuronide. Sulfate conjugates of both testosterone and epitestosterone were also detected in the different urine samples. Glucuronidation assays with the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies UGT1A, UGT2A, and UGT2B revealed that UGT2B17 is the most active enzyme in testosterone glucuronidation. UGT2B17 does not glucuronidate epitestosterone, but inhibition studies revealed that it binds epitestosterone with affinity similar to that of testosterone. Epitestosterone glucuronidation is catalyzed mainly by UGT2B7, and the K(m) of this reaction is significantly lower than the K(m) of UGT2B17 for testosterone. Although UGT2B7 and UGT2B17 exhibited high, although converse, stereoselectivity in testosterone and epitestosterone glucuronidation, UGT2A1, an extrahepatic enzyme that is expressed mainly in the nasal epithelium, catalyzed the glucuronidation of both steroids at considerable rates and similar kinetics. The results shed new light on the substrate specificity and stereoselectivity of human UGTs.

Generic sample preparation and dual polarity liquid chromatography-time-of-flight mass spectrometry for high-throughput screening in doping analysis.

The requirements on initial testing in doping control are getting tighter regarding efficiency and speed while the scope of analytes is getting more diverse and, consequently, the need for high-throughput methods is apparent. In this study, a comprehensive screening method for doping agents in human urine is presented, based on solid phase extraction (SPE) and liquid chromatography-time-of-flight mass spectrometry (LCTOFMS). The method covers most of the compound groups in the list of prohibited substances by World Anti-Doping Agency (WADA). Mixed-mode SPE on two types of sorbent and the use of negative ionization mode besides the commonly used positive mode in electrospray ionization (ESI) allowed detection of acidic compounds, such as sulpho-conjugated metabolites. A run time of 8 minutes for each of the two ESI polarities was achieved. The method was validated regarding relative ionization efficiency, selectivity and signal to noise at the WADA's minimum required performance limit (MRPL) level, resulting in the acceptance of 197 compounds. A selection of 20 compounds was submitted for a more thorough validation, including extraction recovery, repeatability and linearity. Recovery and linearity (R(2)) varied mainly between 83-115% and 0.78-0.99, respectively. Median values for repeatability at the MRPL and 10 x MRPL levels were below 20%. A mean and median mass accuracy of 1.2 and 0.80 mDa, respectively, was achieved. The present method represents at the moment the widest coverage of low molecular weight prohibited substances for the screening in sports, providing an approach for further rationalisation of the analytical work-flow in the doping control laboratories.

Liquid chromatographic-mass spectrometric analysis of glucuronide-conjugated anabolic steroid metabolites: method validation and interlaboratory comparison.

Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine is described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the AAS glucuronides was 89-100% and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts.

Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse-racing.

Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown. In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains.

Aryl-propionamide-derived selective androgen receptor modulators: liquid chromatography-tandem mass spectrometry characterization of the in vitro synthesized metabolites for doping control purposes.

Selective androgen receptor modulators (SARM) are a prominent group of compounds for being misused in sports owing to their advantageous anabolic properties and reduced side effects. To target the preventive doping control analysis in relevant compounds, the challenge is to predict the metabolic fate of a new compound. For aryl-propionamide-derived SARM, an in vitro assay employing microsomal and S9 human liver enzymes was developed to simulate phase-I and phase-II metabolic reactions. In vitro metabolic profiles and the structure-metabolic relationship were compared between four structurally modified substrates. Accurate mass measurements were used to characterize the synthesized metabolites, and also collision-induced dissociation was examined to suggest the methodological approach to monitor the prohibited use of aryl-propionamide-derived drug candidates. Subsequent phase-I and phase-II metabolic reactions were successfully combined in one in vitro assay. The main routes of phase-I modifications involved the hydrolysis of ether linkage, monohydroxylation, and hydrolytic cleavage of the amide bond. Nitro-reduction and deacetylation were reactions observed for substrates possessing the corresponding functionality. SARM metabolites were analyzed in negative ion electrospray ionization and detected as deprotonated species [M-H](-). The main metabolic modifications were observed to occur in the B-ring side, and collision-induced dissociation resulted in the product ions originating from the A-ring side of the compound. These structure-specific ions may be monitored as target ions in the routine doping control.