Effect of glucocorticoid administration on the steroid profile.

The steroid profile (SP) is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids in sports, and it is included in the Athlete Biological Passport (ABP). Glucocorticoids (GCs), which are widely prescribed in sports and only prohibited in competition by systemic routes, inhibit the hypothalamic-pituitary-adrenal axis. Since the metabolites monitored in the SP have a partial adrenal origin, their excretion in urine might be altered by GCs consumption. The aim of the present work was to investigate if GCs administered by either systemic or local routes could influence the SP parameters. Three of the most frequently detected GCs in sports (prednisolone, betamethasone, and triamcinolone acetonide) were administered to healthy male and female volunteers (n=40) using different administration routes (topical, oral, and intramuscular administration at different doses). In total, 66 administrations of GCs were performed. Urine samples were collected before and after GCs administration. The SP was measured using gas chromatography-mass spectrometry. The excretion rates of the SP metabolites decreased after systemic GCs administration. This excretion decrease showed to be associated with the dose and the administration route. However, the individual evaluation of the SP ratios (T/E, A/T, A/Etio, 5αAdiol/5ßAdiol, and 5αAdiol/E) led to normal sequences for all the conditions tested. Therefore, GCs administration did not produce misinterpretations on the ABP evaluation. According to these results, GCs administration should not distort the establishment of normal ranges of the SP ratios, and does not need to be considered a confounding factor in the SP evaluation.

Elimination profile of triamcinolone in urine following oral administration.

Triamcinolone (T) is a glucocorticoid commonly used to relieve inflammation and treat arthritis, severe allergies, and asthma; however, it is banned by the World Anti-Doping Agency in competition for athletes when administered orally, intravenously, intramuscularly, or rectally. The minimum required performance limit (MRPL) for urinary T is 30 ng/mL. However, the data about the urinary excretion of T after oral administration is limited. We investigate the elimination profile and determine whether single-dose administration of T would cause a positive doping result. Twelve healthy volunteers received a single-dose of 4-mg T rally, and urine samples were collected for 24 hours. A validated liquid chromatography-tandem mass spectrometry method was used to determine urinary T levels. Non-compartmental modeling was used to estimate the pharmacokinetic parameters. All the urinary T concentrations were much higher than the MRPL. The peak urinary T concentration was 3211.4 ± 860.3 ng/mL (mean ± SD), time to peak concentration was 1.7 ± 0.9 hours, and the estimated elimination half-life was 4.4 ± 2.8 hours. About 27.76% of the consumed dose was eliminated via urine within 24 hours of intake. After a single-dose oral administration, urinary T concentrations still exceeded the MRPL after 24 hours. This information could be useful for limiting the misuse of T. Athletes should be aware when using T in competition and acquire approval for a therapeutic use exemption prior to use. Moreover, the elimination profile of orally administered T may be crucial information for distinguishing different dosage routes.

Positive doping results caused by the single-dose local injection of triamcinolone acetonide.

Triamcinolone acetonide (TA) is classified as an S9 glucocorticoid in the 2014 Prohibited List published by the World Anti-Doping Agency, which caused it to be prohibited in-competition when administered orally, intravenously, intramuscularly or rectally. The Minimum Required Performance Level (MRPL) for the detection and identification of glucocorticoids is 30 ng/mL. Other common local injection routes, such as intraarticular, intratendinous, or intrabursal injection, are not prohibited. The purpose of this study was to analyze the TA and triamcinolone (T) concentrations in urine after a single injection of TA in patients to determine if it would produce a positive result. This study was performed on 40 patients with sports injuries or joint pains. TA was administered locally (doses varied from 12 to 80 mg). Samples were extracted using a solid-phase extraction column, followed by hydrolysis and liquid extraction using diethyl ether. The elution solvents were collected and dried. The dried residue was reconstituted and assayed by performing liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ionization mode using electrospray ionization and multiple-reaction monitoring as the acquisition mode. The results demonstrated that the concentrations of both TA and T in urine exceeded the MRPL (30 ng/mL) after a single local injection. We obtained positive results for TA in 25 patients, and a positive result for T in one patient. Furthermore, the metabolic situation of TA, a long-acting glucocorticoid, was not an exact linear model. The highest concentrations of TA and T appeared 1-4h after injection. This information could be useful for limiting the misuse of TA by athletes. We suggest that athletes be aware when using TA injections during a competition period and obtain approval for therapeutic use exemption prior to using TA.

A sensitive voltammetric sensor for determination of synthetic corticosteroid triamcinolone, abused for doping.

Edge plane pyrolytic graphite electrode (EPPGE) modified with single-wall carbon nanotubes (SWNTs) has been used as a sensor to determine triamcinolone, abused by athletes for doping. A comparison of the voltammetric behavior between SWNTs modified EPPGE and fullerene - C(60)-modified EPPGE indicated that SWNTs modified EPPGE is more sensitive. The electrode exhibited an effective catalytic response with good reproducibility and stability. The effect of several parameters such as pH, square wave frequency and steroid concentration were studied. The square wave voltammetric response of the electrode to triamcinolone is linear in the range 0.1-25 nM with a detection limit and sensitivity of 8.9 x 10(-10)M and 2.06 microA nM(-1), respectively. The method was applied for the determination of triamcinolone in several commercially available pharmaceuticals and real urine samples obtained from patients undergoing pharmacological treatment with triamcinolone. A comparison of the observed results with HPLC analysis indicated a good agreement. The product obtained after reduction of triamcinolone was also characterized using (1)H NMR and GC-MS and the site of reduction is found to be carbonyl group at position 20. The method described is rapid, simple and accurate and can be easily applied for detecting cases of doping.

Evaluation of different scan methods for the urinary detection of corticosteroid metabolites by liquid chromatography tandem mass spectrometry.

Different approaches for the non-target detection of corticosteroids in urine have been evaluated. As a result of previous studies about the ionization (positive/negative) and fragmentation of corticosteroids, several methods based on both precursor ion (PI) and neutral loss (NL) scans are proposed. The applicability of these methods was checked by the injection of a standard solution containing 19 model compounds. Five of the studied methods (NL of 76 Da; PI of 77, 91 and 105; PI of 237; PI of 121, 147 and 171; and NL of 38 Da) exhibited satisfactory results at the concentration level checked (corresponding to 20 ng/ml in sample). Some other methods in negative ionization mode such as the NL of 104 Da were found to lack sufficient sensitivity. Some of the applied methods were found to be specific for a concrete structure (NL of 38 Da for fluorine containing corticosteroids) while others showed a wide range applicability (PI of 77, 91 and 105 showed response in all model compounds). Interference by endogenous compounds was also tested by the analysis of negative urines and urines spiked with different corticosteroids. The suitability of these methods for the detection of corticosteroid metabolites was checked by the analysis of urine samples collected after the administration of methylprednisolone and triamcinolone. A combination of the reported methods seems to be the approach of choice in order to have a global overview about the excreted corticosteroid metabolites.

New "on-line" sample-pretreatment procedure for routine liquid-chromatographic assay of low-concentration compounds in body fluids, illustrated by triamcinolone assay.

In this fully automated technique for sample cleanup before chromatographic or other quantitation steps, analytes in body fluids are enriched and semi-purified on a first column. After their selective elution, analytes are "transformed" by admixing appropriate solvents in such a way that they are focused on the top of a second column. By backflush, they then are transferred to an analytical liquid-chromatographic column (or simply eluted for quantification by other techniques). This technique is illustrated by the liquid-chromatographic assay of triamcinolone from a 1-mL urine sample, with ultraviolet detection. Because analytical recovery is almost complete and precision high, no internal standardization is necessary. Interference is eliminated as well as or better than with manual techniques. Chief advantages of this technique are online operation, processing of samples of larger volume, low cost with respect to extraction devices, and nearly universal applicability for exogenous or endogenous compounds of clinical relevance. It potentially may be widely applied.