Targeted Metabolite Profiling: Sample Preparation Techniques for GC-MS-Based Steroid Analysis

Many clinical events result in specific and characteristic alterations that originate in biochemical imbalances and are measurable in biological fluids and tissues. Metabolomics consists of the qualitative and quantitative analysis of intraand intercellular metabolites using 2 different analytical methods: (1) non-targeted metabolite profiling and (2)targeted metabolite profiling. Non-targeted metabolite profiling involves the identification and characterization of a large number of metabolites and their precursors. In contrast,targeted metabolite profiling focuses on quantitative changes in metabolites of interest (e.g., amino acids, carbohydrates, steroids, and fatty acids) based on a priori knowledge of the biological function or metabolic pathway. Steroid hormones regulate human body functions and are components of the endocrine system, which coordinates physiological and behavioral responses for specific biological purposes, such as reproduction. Steroids are active at very low concentrations and have a range of biological effects (e.g., homeostasis). Steroid analysis has gained importance in biomedical, pharmaceutical, engineering, and agricultural sciences. Clinical assays for steroid analysis must be robust, highly sensitive, automated, and simple. Steroids analysis is used in the clinical events for the diagnosis of stress,1 Cushing’s syndrome,2 hirsutism,3congenital steroid enzyme deficiency,4 hypertension,5 and as a doping test.6 Biological specimens used in clinical applications include urine, serum, and saliva, and they exhibit some behaviors of steroids (e.g., free forms, conjugated forms, protein-binding forms). Steroids in biological specimens behave differently and different considerations are required when developing methods for their analysis.7 Due to its high sensitivity and selectivity, gas chromatographymass spectrometry (GC-MS) is used to analyze metabolites and precursors of steroid hormones. In recent, GC-MSbased steroid phenol-typing has demonstrated diagnostic power and the potential to discover metabolic diseases.8 This review focuses on the methods involved in a metabolomic study based on GC-MS analysis of steroid hormones in biological specimens. Sample preparation procedures and their major steps are discussed in detail.The various techniques and materials that have been used to achieve efficient analytical results during the past decade are described. Comparative studies of crucial aspects are included to facilitate the understanding of the developed methods.