Method EPA 539 is used for the analysis of hormones. This method detects and quantifies eight different analytes.
The eight analytes tested are:
- 16α-hydroxyestradiol (estriol)
- 17β-estradiol
- 17α-ethynylestradiol
- Testosterone
- Estrone
- 4-androstene-3
- 17-dione
- Equilin
First, the sample is fortified with surrogates. Then it is passed through solid phase extraction (SPE) disks containing octadecyl (C18). This extractd the method analytes and the surrogates.
The compounds are eluted from the solid phase with a small amount of methanol. After this, the extract is concentrated to dryness with the use of nitrogen in a heated water bath.
This is then adjusted to a 1-mL volume with 60:40:methanol after adding the internal standards. The extracts are then 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. This analysis provides a high precise quantitation of these compounds.