I
must admit that as an analytical chemist this is one of my more favorite areas
of chemistry. There are many ways in which to test for steroids i.e. High
Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Gas Chromatography/Mass
Spectrometery (GC/MS), Radio Immuno Assay (RIA) etc. GC/MS is presently the method
of choice for the IOC and most sports federations. Chromatography is a technique
developed by Martin and Synge in England during WWII which netted them the Nobel
prize in Chemistry. It works like this- Imagine mixing sugar, water, iron filings,
and sand together. How would you separate these four components? If you look at
the chemical properties of each they are all different. Iron is magnetic, sand
is inert (doesn't react) and sugar will dissolve in the water. First use a magnet
to pick up the iron filings then pour water over the sand in some filter paper
and evaporate the water out of the filtrate and the sugar is left behind. This
is the same approach we take when mixing chemicals except that we utilize their
different solubilities, and molecular weights. Testosterone weighs 288.41 atomic
mass units (amu). The empirical formula is C19H28O2. Look at any periodic chart
and see that carbon weighs 12 amus, Hydrogen weighs 1 amu and Oxygen weighs 16
amus. Do the math; 19x12=228 + 28x1= 28 +16x2=32=288amus. The extra 0.41 amus
is from isotopic abundances but way too involved to explain.
TestosteroneTestosterone
Cypionate
Testosterone Cypionate weighs 412.59 amus. Its empirical formula is C27H40O3.
Again the math is 12 X27=324, 1x40=40 and 16x3=48 for a total of 412 amus. This
means that in general since Testosterone weighs less it will elute first in a
chromatogram. What do we mean by a chromatogram? The Gas Chromatograph consists
of a hot metal block called an injector, an oven containing a column, to ramp
the temperature, and a detector. Inside the oven resides the column, which looks
like a really long, skinny straw- generally 30 meters (about 98 feet). When steroids
are extracted from urine or some other matrix they are dissolved in some solvent
like methanol. They are injected into the super hot injector (about 300C). The
column does the separating of the chemicals as the temperature of the oven is
ramped because it is coated on the inside with a chemical, which has an affinity
for the steroids. The steroids are pushed through this tiny diameter column by
an inert gas of which helium is most often used. As it comes out the end of the
column it is detected. There are many detectors, which can "see" steroids. Flame
Ionization Detectors (FIDs) and Thermal Conductivity Detectors (TCDs) are commonly
used but only give the retention time as an identification pattern. The most useful
are Mass Spectrometers. Mass Specs are basically a filament (just like the one
in light bulbs) which throw electrons at the steroids causing them to be ionized.
Once ionized they fragment and these fragments are then scanned into a trumpet
shaped device called an electron multiplier, which amplifies the signal. This
signal is then sent to a computer to give its fingerprint fragment pattern, called
a spectrum, which is almost unique to that molecule. This can be compared to a
library of spectra. The following is a chromatogram of 5 steroids from a FID;
One of Paul D. Dodson's wonderful little Anecdotes
Who would have thought that eating candy would be good for you. It's true!
Traditional Chinese medicine cites licorice as a herbal healing agent. Europeans
use it as a soothing agent in cough suppressants and to heal ulcers and in early
western medicine licorice was found to relieve the symptoms of Addison's disease.
Addison's disease is caused by a deficiency of cortisol, a steroid hormone which
among other things helps regulate water retention and blood pressure. Christopher
R. Edwards and Paul M. Stewart from Scotland began to figure out why licorice
was good for treating certain disorders. They suggested that glycyrrhetinic acid,
the steroid constituent of licorice, inhibits an enzyme responsible inactivating
cortisol in the kidney. Eating licorice essentially extends the lifetime of cortisol
in the kidney. (1)
In next months article I will explain the Mass Spectrum of some anabolic steroids.
(1) Chemical and Engineering News, August 12, 2002-Louisa Dalton, Whats that
Stuff pp37.
