Method EPA 539

Method EPA 539 (Hormones) is used for the analysis of hormones. The analytes that are detected and quantified by this method are 16α-hydroxyestradiol (estriol), 17β-estradiol, 17α-ethynylestradiol, testosterone, estrone, 4-androstene-3, 17-dione and equilin.

The sample is fortified with surrogates and passed through solid phase extraction (SPE) disks containing octadecyl (C18) functional groups in order to extract the method analytes and the surrogates. The compounds are eluted from the solid phase with a small amount of methanol. The extract is concentrated to dryness with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with 60:40 methanol: after adding the internal standards. The extracts are is injected into liquid chromatograph where the analytes are separated based on polarity and passed into two mass spectrometer in tandem for identification based on the molecular masses of the fragmented compounds. In addition, this analysis provides a high precise quantitation of these compounds.