I have been searching google and didn't found any links that describes how this process is performed - I mean the chemical procedure, not the process of taking the blood from the vein.


The steps for hormone level measurement generally follow a similar sequence. For the most accurate analysis liquid chromatography-mass spectroscopy (lc-ms) is typically used. The liquid chromatography separates the sample into fractions, conditions are selected such that a clean, separate peak is seen for the hormone being evaluated. The purified hormone derived from the liquid chromatography step is then introduced into the mass spectrometer for quantification.

The general process involves:

  • sample collection: a sample of saliva, blood or urine is collected
  • addition of a known quantity of hormone to act as an internal standard. Often a $\ce{^{13}C}$ labeled hormone is used (commercially available). The parent ion for the labeled standard will occur one mass unit higher than the parent ion for naturally occurring $\ce{^{12}C}$-containing collected sample. If the peak height of the sample in the mass spec is half as large as that of the standard, then the concentration of hormone in the collected sample is half that in the standard.
  • chemically separate the hormone from any large binding proteins
  • isolate the fraction containing the hormone using liquid-liquid extraction
  • run a second liquid-liquid extraction to remove highly polar materials
  • run lc-ms analysis on sample that has now been purified and enriched in hormone

Here is a link to a detailed procedure for testosterone analysis.

To keep things understandable I have simplified the description of some of the steps in the process. For example lc-tandem mass spectrometry is often used to improve accuracy.

Research today in hormone quantification is focused on reducing cost and turn-around time. A significant amount of work is particularly being done on analyzing the raw collected sample without further purification.

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