An analytical method based on on-line SPE-LC-APCI-MS/MS has been developed for the detection and quantification of eight selected estrogenic and progestagenic steroid hormones; estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), levonorgestrel (LEVO), medroxyprogesterone (MEDRO), norethindrone (NORE) and progesterone (PROG) in wastewater matrices. The injection volume could range from 1 to 10-mL according to the expected concentration of steroid hormones in matrix. The method characteristics are: analysis time per sample (<15 min), acceptable recovery values (71-95%), good precision (RSD ≤ 10%) and limits of detection at the low-nanogram per liter levels in affluent and effluent wastewaters (8-60 ng L(-1)). In particular, a detailed discussion of optimization parameters impacting overall performance of the method has been presented (sample collection, filtration and storage). All optimization and validation experiments for the on-line SPE method and chromatographic separation were performed in environmentally-relevant wastewater matrices. This method represents a compromise between analysis time, higher sample throughput capabilities, sample volume and simplicity for the analysis of both progestagenic and estrogenic steroid hormones in a single run, with LODs and LOQs sufficiently low to detect and quantify them in environmental wastewater matrices. Thus, the applicability of the method was tested on affluent and effluent wastewaters from two wastewater treatment facilities using different processes (biological and physico-chemical) to evaluate their removal efficiency for the detected steroid hormones.
Some of the approved drugs are synthetic versions of the natural hormones, such as trenbolone acetate and zeranol. Just like the natural hormone implants, before FDA approved these drugs, FDA required information and/or toxicological testing in laboratory animals to determine safe levels in the animal products that we eat (edible tissues). Furthermore, FDA required that the manufacturers demonstrate that the amount of hormone left in each edible tissue after treatment is below the appropriate safe level. As described above, a safe level is a level which would be expected to have no harmful effect in humans.
Because steroids are lipophilic, they diffuse easily through the cell membranes, and therefore have a very large distribution volume. In their target tissues, steroids are concentrated by an uptake mechanism which relies on their binding to intracellular proteins (or " receptors ", see below). High concentration of steroids are also found in adipose tissue, although this is not a target for hormone action. In the human male, adipose tissue contains aromatase activity, and seems to be the main source of androgen-derived estrogens found in the circulation. But most of the peripheral metabolism occurs in the liver and to some extent in the kidneys, which are the major sites of hormone inactivation and elimination, or catabolism (see below).