A homogeneous sol particle immunoassay for total oestrogens in urine and serum samples

Chromatography as a reference technique for the determination of clinically important steroids

“…Preferred solvents for extracting nonpolar, hydrophobic steroids (e.g., androgens, oestrogens) are diethylether or benzene (Cook et al, 1982;Feher et al, 1977;Kling and diZerega, 1982;Moneti et al, 1987) whereas more polar solvents (dichloromethane, chloroform, ethylacetate) are useful for a broader range of steroids including corticosteroids (Allenmark ei al., 1981;Barron et al, 1979;HalperinWalega and Greene, 1985;Wiebe et al, 1980). The recovery of polar steroids and steroid conjugates is improved by addition of inorganic salts (e.g., sodium chloride, ammonium sulphate) to samples (Barron et al, 1979;Leuvering et al, 1983). Moreover, the extraction of nonsteroidal lipids from plasma is minimized by the addition of sodium hydroxide prior to extraction (Ando et al, 1986;Makin, 1975).…”

“…In cases where immediate analysis of samples is either impractical or impossible, unstabilised biological fluids may be stored overnight at 2 -8 "C. For longer periods, storage at -20 "C in airtight containers is adequate (Leuvering et al, 1983;Reiffsteck et al, 1982;Risk et al, 1984;Fotsis, 1987) although lower temperatures (Ando et al, 1986) have also been used. Nonpolar steroids (eg.…”

“…In contrast, 24 h urine specimens are collected in the presence of a preservative such as boric acid (Barron et al, 1979) or ascorbic acid (Fotsis, 1987) to prevent bacterial degradation of steroids and hydrolysis of steroid conjugates. Although inconvenient to collect, analysis of a 24 h urine specimen (Fotsis, 1987;Leuvering et al, 1983;Barron et al, 1979) represents the average steroid excretion over 24 h thus avoiding the problem of circadian variation of steroid concentrations. Salivary sampling regimens, on the other hand, have the advantage of frequent, easy collection by noninvasive techniques which obviate difficulties of ensuring completeness of a 24 h urine collection.…”

“…Caution must be exercised in the handling of samples since repeated freezing and thawing can cause hydrolysis of conjugates and yield false high values for unconjugated steroids. Thawed specimens are frequently centrifuged to remove precipitates (Leuvering et al, 1983) including precipitated protein (Andreolini et al, 1987;Moneti et a l , 1987).…”

“…Two general procedures exist for the hydrolysis of conjugates. In acid hydrolysis, the sample is boiled (Leuvering et al, 1983), usually under reflux, in the presence of a mineral acid for a specified time (10 -60 min). For enzymatic hydrolysis (Barron et al, 1979;Cartoni and Coccioli, 1983;Beyssac et al, 1986;Purdy et al, 1980;Reiffsteck et al, 1982;Risk et al, 1984), the sample is buffered to the optimal pH for the enzyme and after addition of sulphatase and/or glucuronidase is incubated for 18 -72 h at, for example, 37 "C. These mild conditions minimize steroid degradation during hydrolysis, but the possible presence of enzyme inhibitors (Fotsis and Adlercreutz, 1987) casts some doubt on the completeness of hydrolysis (Kuelpmann and Breuer, 1977).…”