This Test Guideline describes an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line, used for the assay, expresses genes that encode for all the key enzymes for steroidogenesis. After an acclimation period of 24 h in multi-well plates, cells are exposed for 48 h to seven concentrations of the test chemical in at least triplicate. Solvent and a known inhibitor and inducer of hormone production are run at a fixed concentration as negative and positive controls. At the end of the exposure period, cell viability in each well is analyzed. Concentrations of hormones in the medium can be measured using a variety of methods including commercially available hormone measurement kits and/or instrumental techniques such as liquid chromatography-mass spectrometry. Data are expressed as fold change relative to the solvent control and the Lowest-Observed-Effect-Concentration. If the assay is negative, the highest concentration tested is reported as the No-Observed-Effect-Concentration.
Steroid isolation , depending on context, is the isolation of chemical matter required for chemical structure elucidation, derivitzation or degradation chemistry, biological testing, and other research needs (generally milligrams to grams, but often more  or the isolation of "analytical quantities" of the substance of interest (where the focus is on identifying and quantifying the substance (for example, in biological tissue or fluid). The amount isolated depends on the analytical method, but is generally less than one microgram.  [ page needed ] The methods of isolation to achieve the two scales of product are distinct, but include extraction , precipitation, adsorption , chromatography , and crystallization . In both cases, the isolated substance is purified to chemical homogeneity; combined separation and analytical methods, such as LC-MS , are chosen to be "orthogonal"—achieving their separations based on distinct modes of interaction between substance and isolating matrix—to detect a single species in the pure sample. Structure determination refers to the methods to determine the chemical structure of an isolated pure steroid, using an evolving array of chemical and physical methods which have included NMR and small-molecule crystallography .  : 10–19 Methods of analysis overlap both of the above areas, emphasizing analytical methods to determining if a steroid is present in a mixture and determining its quantity.