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. 
In addition to their characteristic hydroxylase activities, some steroidogenic CYP enzymes also catalyse lyase reactions. Specifically, the CYP11A (cytochrome P450 cholesterol side chain cleavage) and CYP17 (cytochrome P450 17α-hydroxylase/C17,20-lyase) enzymes can each catalyse cleavage of the C․C bond weakened by the hydroxylation reactions. CYP11A acts in the IMM to catalyse the rate-limiting reaction in steroid synthesis: the conversion of cholesterol to pregnenolone. This crucial CYP enzyme hydroxylates two adjacent carbons (C20 and C22) in the D-ring side chain of cholesterol, facilitating cleavage between C20 and C22 to leave the Δ 5 21 carbon steroid, pregnenolone ( Miller 2008 ). Similarly, CYP17 introduces a hydroxyl group at position C17 of either pregnenolone or progesterone, as a result of which the weakened C17–C20 bond breaks to generate either DHEA or androstenedione, respectively ( Miller 2008 ) (see Figure ). Other members of the CYP enzyme family simply catalyse introduction of oxygen to generate hydroxyl groups at specific carbon positions (see Table ). The hydroxylations catalysed by CYP21 (21-hydroxylase) and CYP11B1 (11β-hydroxylase) are pivotal in the formation of corticosteroids by the adrenal cortex ( Miller 2008 ). The hydroxyl group generated at position C18 by CYP11B2 (aldosterone synthase) undergoes rapid oxidation to form an aldehyde group ( Curnow et al 1991 ), hence giving rise to the name ‘aldosterone’ (see Figure ). Finally, CYP19 (aromatase) is the most complex member of the steroidogenic CYP enzyme family, catalysing a series of reactions that convert C19 androgens (androstenedione and testosterone) to their C18 oestrogen metabolites (oestrone and oestradiol, respectively). In this reaction sequence, the C19 methyl group is lost and the ketone at position C3 is reduced to a hydroxyl group ( Miller 2008 ). This liberates electrons which are invested in the A-ring of the steroid to generate the aromatic phenol ring, the hallmark of oestrogens (see Figure ) and a prerequisite for activation of the oestrogen receptor.