Drug Testing and Analysis

Gene expression profiling might be a suitable technique for doping analysis. But to avoid false positive interpretation several aspects have to be taken into account. Exercise, circadian rhythm and hormone application as well as blood sampling could modulate gene expression results.

RNA interference leads to the modulation of gene expression levels and antisense oligonucleotides, siRNA molecules as well as antagomirs could be used for performance enhancement. This review gives an overview on the structure of the molecules, their degradation in vivo and in vitro and detection strategies.

In accord with the idea that doping substances influence mRNA expression profiles, it has been suggested that these changes can be detectable by screening the blood transcriptome. After almost one decade of research using transcriptomic tools, it still remains a matter of future technological progress to identify the ultimate biomarker using technologies and/ or methodologies that are sufficiently robust against typical biological or technical bias and valid in a court of law.

This manuscript describes the efforts made in our lab concerning gene expression analysis in different species and tissues after treatment with anabolic steroid hormones in order to find biomarkers to screen for the abuse of anabolic steroid hormones in human sports and animal husbandry.

The review focuses on the relationship between epigenetics and sports, highlights the potential consequences of abuse of doping drugs on gene expression and describes methods to detect epigenetic changes of gene markers reflecting the physiological or metabolic effects of doping agents. Basics of DNA methylation and histone modifications and their relationship to nutrition and sport are described. The potential effects of long-term intakes of growth hormone and IGF-1 on epigenetic mechanisms along with an increased risk of cancer are discussed.

Following lorazepam administration (2 mg, oral) to volunteers, metabolites were identified in urine by LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone and particularly the quinazoline carboxylic acid provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis to rapidly reduce the time spent on analysis.

Results of in vitro experiments to elucidate the equine metabolism of eight previously unstudied sedatives are presented. The use of high-resolution accurate-mass LC-MS along with in vitro methods provide a means of detailing the metabolism of these sedatives, which are not licensed for use in horses in the UK.

Mesocarb that is the central nervous system stimulant are studied, and horse doping control detection method based on liquid chromatography-tandem mass spectrometry is presented.

Two horses were treated with sildenafil, and its metabolic products were sought in both urine and plasma samples. Prior to this, a simulative laboratory study had been done using the photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in the biological samples. Several products were formed and characterized using the HPLC/HRMSⁿ technique.

Acamprosate was estimated from human plasma by ESI-LC-MS/MS. This is the most sensitive method with very short analysis time. The linearity range of acamprosate is 7.04–702.20 ng/mL. Total analysis time is 2.5 minutes only. It shows remarkable matrix effects in Z-spray ion source design, but almost no matrix effects in orthogonal spray ion source design.

The proposed electrochemical behaviour can be successfully applied for the determination of tamoxifen in pharmaceutical formulation. The peak current is found to be linear over the concentration range 1.0–5.0 (SWV) and 1.0–6.0 µgmL⁻¹ (DPV). The procedure has been applied to the assay of the drug in tablets form with mean percentage recoveries of 99.98%.

A sensitive and selective spectrophotometric method for the assay of gabapentin (GBP) in capsules was developed. The method is based on the reaction of GBP through its amino group with sodium 1, 2-naphthoquinone -4-sulfonate in the presence of Clark and Lubs buffer of pH 11.0.