A rapid and eco-friendly method for determination of the main components of gamma-oryzanol in equestrian dietary and nutritional supplements by liquid chromatography-Tandem mass spectrometry.

Gamma-oryzanol (GO) has gained special attention in the equine sports industry in recent years due to its touted properties, including the fact that it may cause anabolic effects on muscle growth and reduce fatigue. Many manufactures offer supplements containing GO as a naturally occurring anabolic substance; however, some producers do not declare its presence in product compositions. Taking into consideration the touted properties of GO, its ambiguous effectiveness and the open character of the Prohibited Substances List established by the Fédération Equestre Internationale, there is an urgent need to elaborate procedures for the estimation of horse exposure to GO during supplementation, as well as during routine analysis of supplements. This work describes the development and validation of the method for determination of the four main GO components, i.e., cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate and ß-sitosteryl ferulate, in equestrian supplements based on LC-MS/MS after a simple ultrasound-assisted extraction (Eco-Scale score value of 76). The analytical performance achieved satisfactory results in terms of linearity (R2 > 0.9910), sensitivity (LODs ranged from 0.4 to 1.9 ng/mL), intra- and interday accuracy (from 90.4-115.8%), precision (CV < 9.6%) and recovery (from 87.6-108.6%) for all of the investigated compounds. The method was successfully applied to the analysis of thirty equestrian supplements.

Development of an extraction method for anabolic androgenic steroids in dietary supplements and analysis by gas chromatography-mass spectrometry: Application for doping-control.

Several studies have highlighted that nutritional supplements may contain undeclared anabolic steroids that are banned by the International Olympic Committee/World Anti-Doping Agency. Any kind of abuse with these drugs is extremely dangerous because of their side effects. Thus, the control of food additives in order to protect the best consumer health and to limit fraudulent practices in the field of sports is essential. This paper describes a simple and effective qualitative gas chromatography-mass spectrometry (GC-MS) method to detect anabolic androgenic steroids (AAS): androsterone, nandrolene, dehydroepiandrosterone, 5ɑ-androstane-3ß, 17ß-diol, dihydrotestosterone, testosterone, methenolone acetate, methandienone, boldenone and fluoxymesterone, in food supplements. Methyltestosterone was used as internal standard. Target compounds were extracted with a mixture of N-pentane and di-ethylether (7.5:2.5, v/v). A good extraction recovery was obtained by our method for all the AAS (R > 88%). Crude extract was derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Separation was performed on a GC connected to quadrupole MS detector using a 5% phenylmethylsiloxane fused silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 µm). Helium was used as carrier gas with a flow rate of 0.3 µl min-1 (measured at 6.1 psi 190 °C). The MS was operated in electron ionization mode (70 eV) and in selected ion monitoring (SIM). The mass spectra of the standard compounds were acquired in full SCAN mode (50-700 m/z) by infusion of a reference solution at 50 µg/ml. Three higher diagnostic ions were monitored for each compound of interest. All AAS get separated with good peak shapes and resolution factor. The total analysis time by our optimised method was only 20 min. The developed method was validated according to Laboratories International Standard regulations for specificity, precision in both liquid and solid matrixes, and memory effect. The Tolerance Interval was judged true. The limit of detection was about 10 ng/g for solid samples and 10 ng/ml for liquid samples. The developed method was then applied to the research of steroids in nine Tunisian commercially dietary supplements using for each compound of interest SIM mode for screening then SCAN mode for confirmation. One of the monitoring samples was positive to methandienone not declared on the label. Our analytical method can be beneficial for AAS screening in dietary supplements.

Hepatotoxicity by Dietary Supplements: A Tabular Listing and Clinical Characteristics.

Dietary supplements (DS) are extensively consumed worldwide despite unproven efficacy. The true incidence of DS-induced liver injury (DSILI) is unknown but is probably under-diagnosed due to the general belief of safety of these products. Reported cases of herbals and DS-induced liver injury are increasing worldwide. The aim of this manuscript is to report a tabular listing with a description of DS associated with hepatotoxicity as well as review the phenotype and severity of DSILI. Natural remedies related to hepatotoxicity can be divided into herbal product-induced liver injury and DS-induced liver injury. In this article, we describe different DS associated with liver injury, some of them manufactured DS containing several ingredients (Herbalife™ products, Hydroxycut™, LipoKinetix™, UCP-1 and OxyELITE™) while others have a single ingredient (green tea extract, linoleic acid, usnic acid, 1,3-Dimethylamylamine, vitamin A, Garcinia cambogia and ma huang). Additional DS containing some of the aforementioned ingredients implicated in liver injury are also covered. We have also included illicit androgenic anabolic steroids for bodybuilding in this work, as they are frequently sold under the denomination of DS despite being conventional drugs.

Synthetic androgens as designer supplements.

Anabolic androgenic steroids (AAS) are some of the most common performance enhancing drugs (PED) among society. Despite the broad spectrum of adverse effects and legal consequences, AAS are illicitly marketed and distributed in many countries. To circumvent existing laws, the chemical structure of AAS is modified and these designer steroids are sold as nutritional supplements mainly over the Internet. Several side effects are linked with AAS abuse. Only little is known about the pharmacological effects and metabolism of unapproved steroids due to the absence of clinical studies. The large number of designer steroid findings in dietary supplements and the detection of new compounds combined with legal loopholes for their distribution in many countries show that stricter regulations and better information policy are needed.

Effects of aromatase inhibitors on human osteoblast and osteoblast-like cells: a possible androgenic bone protective effects induced by exemestane.

Effects of aromatase inhibitors (AIs) on the human skeletal system due to systemic estrogen depletion are becoming clinically important due to their increasing use as an adjuvant therapy in postmenopausal women with breast cancer. However, possible effects of AIs on human bone cells have remained largely unknown. We therefore studied effects of AIs including the steroidal AI, exemestane (EXE), and non-steroidal AIs, Aromatase Inhibitor I (AI-I) and aminoglutethimide (AGM), on a human osteoblast. We employed a human osteoblast cell line, hFOB, which maintains relatively physiological status of estrogen and androgen pathways of human osteoblasts, i.e., expression of aromatase, androgen receptor (AR), and estrogen receptor (ER) beta. We also employed osteoblast-like cell lines, Saos-2 and MG-63 which expressed aromatase, AR, and ERalpha/beta in order to further evaluate the mechanisms of effects of AIs on osteoblasts. There was a significant increment in the number of the cells following 72 h treatment with EXE in hFOB and Saos-2 but not in MG-63, in which the level of AR mRNA was lower than that in hFOB and Saos-2. Alkaline phosphatase activity was also increased by EXE treatment in hFOB and Saos-2. Pretreatment with the AR blocker, flutamide, partially inhibited the effect of EXE. AI-I exerted no effects on osteoblast cell proliferation and AGM diminished the number of the cells. hFOB converted androstenedione into E2 and testosterone (TST). Both EXE and AI-I decreased E2 level and increased TST level. In a microarray analysis, gene profile patterns following treatment with EXE demonstrated similar patterns as with DHT but not with E2 treatment. The genes induced by EXE treatment were related to cell proliferation, differentiation which includes genes encoding cytoskeleton proteins. We also examined the expression levels of these genes using quantitative RT-PCR in hFOB and Saos-2 treated with EXE and DHT and with/without flutamide. HOXD11 gene known as bone morphogenesis factor and osteoblast growth-related genes were induced by EXE treatment as well as DHT treatment in both hFOB and Saos-2. These results indicated that the steroidal aromatase inhibitor, EXE, stimulated hFOB cell proliferation via both AR dependent and independent pathways.