Electrophoretic Determination of Trimethylamine (TMA) in Biological Samples as a Novel Potential Biomarker of Cardiovascular Diseases Methodological Approach.

In competitive athletes, the differential diagnosis between nonpathological changes in cardiac morphology associated with training (commonly referred to as "athlete's heart") and certain cardiac diseases with the potential for sudden death is an important and not uncommon clinical problem. The use of noninvasive, fast, and cheap analytical techniques can help in making diagnostic differentiation and planning subsequent clinical strategies. Recent studies have demonstrated the role of gut microbiota and their metabolites in the onset and the development of cardiovascular diseases. Trimethylamine (TMA), a gut bacteria metabolite consisting of carnitine and choline, has recently emerged as a potentially toxic molecule to the circulatory system. The present work aims to develop a simple and cost-effective capillary electrophoresis-based method for the determination of TMA in biological samples. Analytical characteristics of the proposed method were evaluated through the study of its linearity (R2 > 0.9950) and the limit of detection and quantification (LOD = 1.2 µg/mL; LOQ = 3.6 µg/mL). The method shows great potential in high-throughput screening applications for TMA analysis in biological samples as a novel potential biomarker of cardiovascular diseases. The proposed electrophoretic method for the determination of TMA in biological samples from patients with cardiac disease is now in progress.

Determination of bromhexine and its metabolites in equine serum samples by liquid chromatography - Tandem mass spectrometry: Applicability to the elimination study after single oral dose.

Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneous determination of BH and its metabolites, including 4-(2-amino-3,5-dibromobenzylamino)cyclohexanol (4-HDMB), 3-(2-amino-3,5-dibromobenzylamino)cyclohexanol (3-HDMB), in equine serum samples. The 2-(2-amino-3,5-dibromobenzylamino)cyclohexanol (2-HDMB) was monitored as well. The assay was validated in terms of linearity (R2 greater than 0.9951), intra- and inter-assay accuracy (91.6 - 109.1%) and precision (CV < 9.6%) as well as recovery (94.8 - 105.65%). The LODs were 0.0052, 0.0053, 0.0056 and 0.0043 ng/mL for BH, 2-HDMB, 3-HDMB and 4-HDMB, respectively. The developed method was applied to determine the time curses of BH and its metabolites concentrations in equine serum collected for 95.25 h following a single oral administration of BH to two healthy mares (in dose of 0.8 mg/kg). The parent drug was found at higher concentration levels than 3-HDMB (major metabolite) and 4-HDMB (minor metabolite), however, both BH metabolites lasted for a longer period of time in equine serum than the parent drug. Thus, both metabolites of BH can be considered as BH abuse markers.

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.

Application of micellar electrokinetic chromatography for detection of silver nanoparticles released from wound dressing.

The recent emergence of nanotechnology has provided a new therapeutic modality in case of silver nanoparticles. Dressings containing silver form the basis for the treatment of burns and wounds, either acute or chronic ones. The aim of the study was to examine silver release from the different wound dressings: commercially available (Atrauman Ag, Aquacel Ag) and experimental (FKDP-AgNPs) using MEKC. In order to characterize prepared keratin based wound dressing before and after its modification with AgNPs, a compositional analysis was conducted using energy dispersive X-ray spectroscopy. Nanosilver toxicity was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium test. Silver release from wound dressings was assessed using MEKC. The best separation was observed for MEKC in 20 mM borate buffer at pH 9 with 20 mM SDS addition. In vitro studies showed silver at higher concentration than 10 ppm exerted a toxic effect on fibroblasts isolated from diabetic mice versus. NIH/3T3 and BJ cell lines (p < 0.05). We observed silver was released more gradually from experimental FKDP-AgNPs wound dressing, in compare to commercially available wound dressings. The fast and low-cost method utilizing MEKC can be used in clinical practice to detect silver release from the wound dressings.

Comparison of GC-MS and MEKC methods for caffeine determination in preworkout supplements.

In this study, GC-MS- and MEKC-based methods for determination of caffeine (CAF) in preworkout supplements were developed and validated. The proposed protocols utilized minimal sample preparation (simple dilution and syringe filtration). The developed methods achieved satisfactory validation parameters, i.e. good linearity (R2 > 0.9988 and R2 > 0.9985 for GC-MS- and MEKC-based method, respectively), satisfactory intra- and interaccuracy (within 92.6-100.7% for method utilizing GC-MS and 92.1-110.3% for protocol based on MEKC) and precision (CV < 15.9% and CV < 6.3% for GC-MS- and MEKC-based method, respectively) and recovery (within 100.1-100.8% for method utilizing GC-MS and 101.5-106.2% for protocol based on MEKC). The LOD was 0.03 and 3 µg/mL for method utilizing GC-MS and MEKC, respectively. The CAF concentrations determined by GC-MS- and MEKC-based methods were found to be in the range of 8.53-11.23 and 8.20-11.61 µg/mL, respectively. Taking into consideration information on the labels, the investigated supplements were found to contain from 110.0 to 167.3% of the declared CAF content, which confirmed the literature reports on incompatibility of the declared product compositions with real ones. Nevertheless, the consumption of examined supplements as recommended by producers did not lead to exceeding the CAF safe limit of 400 mg per day. Additionally, the MEKC-based method allowed for detection and identification of vitamin B3 and B6 in all of the investigated supplement samples, which demonstrated that MEKC-based protocols may be an appropriate assays for simultaneous determination of CAF and vitamins.

A rapid and sensitive method for the quantitative analysis of ibuprofen and its metabolites in equine urine samples by gas chromatography with tandem mass spectrometry.

Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 µL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optimized and validated according to the principles for bioanalytical methods. The assay achieved satisfactory validation parameters, namely, recovery (92.2-105%), interday accuracy (92.5-106%), and precision (0.3-4.4%) for all investigated compounds as well as limits of quantification of 50 ng/mL for ibuprofen, 2-hydroxyibuprofen, and carboxyibuprofen, 25 ng/mL for 1-hydroxyibuprofen and 100 ng/mL for 3-hydroxyibuprofen. The applicability of the method was evaluated by the analysis of five real urine samples collected from different horses after drug administration. In view of the low limits of quantification, high selectivity, repeatability, and recovery, the procedure can be utilized for laboratory applications, including the control of ibuprofen abuse in equestrian sports for anti-doping purposes and drug/pharmaceutical mentality investigations.

Simultaneous determination of ibuprofen and its metabolites in complex equine urine matrices by GC-EI-MS in excretion study in view of doping control.

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 µg mL-1, 0.62 µg mL-1, 4.15 µg mL-1, 0.58 µg mL-1 and 4.04 µg mL-1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial - the potential of enantiomeric separation for doping control analysis.

The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes' claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed.