ABSTRACT
In sterol analysis two internal standards are frequently used: β-cholestanol and 5αcholestane. β-cholestanol is structurally very similar to the sterols and is hence a perfect internal standard for samples void of stanols that are analyzed using a high-resolution GC method (see, for example, Fig. 7). It cannot be used in combination with low resolution GC methods because on such systems it will coelute with cholesterol. Its use is also impossible if stanol mixtures are to be analyzed. Such mixtures are very likely to contain significant levels of the compound as a result of cholesterol hydrogenation. An alternative internal standard finally also is needed if low levels of sterols have to be quantified in samples that contain a very high level of cholesterol, such as blood samples. For such samples the separation of the β-cholestanol internal standard from the cholesterol is difficult at best. If β-cholestanol, the internal standard of first choice, cannot be used, 5α-
Cholestane can be a reasonable alternative. Although this compound is structurally similar to the sterols, the lack of the hydroxy group on position 3 in the A-ring can make it chemically very different from the sterols or stanols. As a result of this it cannot, for example, be used in NPLC class type separations where it will elute significantly earlier than all sterols. In addition to the two internal standards mentioned above, alternative compounds such as betulin, are occasionally used (45). In contrast to the sterols and
stanols this compound has two hydroxy groups, which means that it will behave slightly different from the normal sterols. Moreover, its FID response factor will be slightly different, both in the underivatized and in the silylated form. For sterol oxide analysis, other model compounds are evidently needed. A good candidate here is 19-OH cholesterol (46).
