Page 61 - CRFTApplication Compendium_090613

Quantitative Analysis of Testosterone in Serum
by LC-MS/MS
Ravinder J. Singh, Ph.D., James L. Bruton, Mayo Clinic, Rochester, MN, USA
Taha Rezai, Ph.D., Thermo Fisher Scientific, San Jose, CA, USA
Introduction
Testosterone is the major androgenic hormone. It is
responsible for the development of the male external
genitalia and secondary sexual characteristics. In females,
its main role is as an estrogen precursor. In both genders,
it also exerts anabolic effects and influences behavior.
In men, testosterone is secreted by the testicular Leydig
cells and, to a minor extent, by the adrenal cortex. In
premenopausal women, the ovaries are the main source of
testosterone with minor contributions by the adrenals and
peripheral tissues. After menopause, ovarian testosterone
production is significantly diminished. Testosterone
production in testes and ovaries is regulated via pituitary-
gonadal feedback involving luteinizing hormone (LH) and,
to a lesser degree, inhibins and activins.
Most circulating testosterone is bound to sex hormone-
binding globulin (SHBG), which in men also is called
testosterone-binding globulin. A lesser fraction is albumin
bound and a small proportion exists as free hormone.
Historically, only the free testosterone was thought to be
the biologically active component. However, testosterone
is weakly bound to serum albumin and dissociates freely
in the capillary bed, thereby becoming readily available
for tissue uptake. All non-SHBG-bound testosterone is
therefore considered bioavailable.
For adults, the normal values for testosterone are
240-950 ng/dL for males and 8-60 ng/dL for females.
Goal
To develop a sensitive, quantitative LC-MS/MS assay for
testosterone in serum for research laboratories.
Experimental Conditions/Methods:
Chemicals and Reagents
Testosterone standard was purchased from Steraloids, Inc.
in the powder form and is stored at room temperature.
The internal standard, Testosterone 16,16,17-d3, was
purchased from CDN Isotopes in the powder form and is
also stored at room temperature. Bovine serum albumin
and PBS buffer were purchased from Sigma-Aldrich and
stored in a refrigerator. Bovine serum was used because
human serum with undetectable levels of testosterone
was not commercially available.
Sample Preparation
0.025 mL deuterated stable isotope internal standard
(d3 -testosterone) is added to a 0.1 mL serum sample as
internal standard. Protein is precipitated from the mixture
by the addition of 0.25 mL acetonitrile. The testosterone
and internal standard are extracted from the resulting
supernatant by an on line extraction. This is followed by
conventional liquid chromatography and analysis on a
tandem mass spectrometer equipped with a heated
nebulizer ion source.
Calibration Curve Standards Preparation
A standard stock solution of 1 mg/mL of testosterone
was prepared in methanol. Standard spiking solutions
of testosterone in methanol/water at concentrations of
1000 ng/mL and 100 ng/mL were prepared by dilution
of the stock standard solution. The appropriate amount
of standard spiking solution was added to 100 mL of
5% BSA in 0.01M PBS (pH 7.4) to prepare calibration
standards at the following concentrations: 5 ng/dL, 10 ng/dL,
20 ng/dL, 50 ng/dL, 100 ng/dL, 200 ng/dL, 500 ng/dL,
1000 ng/dL, and 2000 ng/dL. The standards were
processed with the sample preparation procedure
described above. The standard stock solution and
the standard spiking solutions were stored at -20 °C.
HPLC
HPLC analysis was performed using the Thermo Scientific
Transcend TLX-2 System. The 0.1 mL samples were
injected onto a 4 x 2 mm C18 Guard cartridge that served
as an extraction column. The analyte was directly
transferred from the extraction column and focused onto
the 33 x 4.6 mm analytical column which was packed
with 3 micron particles. Loading and Eluting Mobile
phase A was water. Loading phase B was methanol.
Loading phase C was a solution containing 45% acetonitrile,
45% isopropanol, and 10% acetone which is used to wash
the extraction column. Eluting Mobile phase B was a 50/50
solution of water and acetonitrile. The appropriate
gradients and flow rates are described in Table 1.
MS/MS
MS/MS analysis was carried out on a Thermo Scientific
TSQ Quantum Ultra triple stage quadrupole mass
spectrometer with an atmospheric pressure chemical
ionization (APCI) probe.
Key Words
• TSQ Quantum
Ultra
• Clinical Research
• LC-MS/MS
• Transcend TLX-2
Application
Note: 429
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