67 / 658
Quantitative Analysis of Cortisol and Cortisone
in Urine by LC-MS/MS
Ravinder J. Singh, Ph.D., James L. Bruton, Mayo Clinic, Rochester, MN
Introduction
Cortisol is a steroid-hormone synthesized from cholesterol by
a multienzyme cascade in the adrenal glands. It is the main
glucocorticoid in humans and acts as a gene-transcription
factor influencing a multitude of cellular responses in
virtually all tissues. Its production is under hypothalamic-
pituitary feedback control.
Only a small percentage of circulating cortisol is
biologically active (free), with the majority of cortisol
inactive (protein bound). As plasma cortisol values
increase, free cortisol (i.e., unconjugated cortisol and
hydrocortisone) increases and is filtered through the
glomerulus. Urinary free cortisol (UFC) in the urine
correlates well with the concentration of plasma free
cortisol. UFC represents excretion of the circulating,
biologically active, free cortisol.
Goal
To develop a sensitive quantitative LC-MS/MS method for
measuring cortisol and cortisone in urine for research
applications.
Experimental Conditions/Methods
Chemicals and Reagents
Cortisol standard was purchased from the National Bureau
of Reference Materials in the powder form and is stored
at room temperature. Cortisone standard was purchased
from Sigma in the powder form and is stored at room
temperature. The internal standard, Cortisol 9,12,12-d
3
,
was purchased from Cambridge Isotope Laboratory in the
powder form and is also stored at room temperature.
Stripped urine was purchased from SeraCare Life Sciences
and is stored at -20 °C.
Sample Preparation
0.050 mL deuterated stable isotope (d3-cortisol) is added
to a 0.1 mL urine sample as internal standard. The
cortisol, cortisone and internal standard are extracted by
an online extraction utilizing high-throughput liquid
chromatography (HTLC). 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 cortisol and
cortisone was prepared in methanol. Standard spiking
solutions of cortisol and cortisone in methanol/water at
concentrations of 5 µg/mL were prepared by dilution of
the stock standard solution. The appropriate amount of
standard spiking solution was added to 100 mL of
stripped urine to prepare calibration standards at the
following concentrations: 0.25 µg/dL, 1 µg/dL, 4 µg/dL,
and 20 µg/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
Aria TLX-2 System. The 0.1 mL samples were injected onto
a Thermo Scientific 0.5 x 50 mm C18 HTLC Column that
served as an extraction column. The analyte was directly
transferred from the extraction column and focused onto
the analytical column which was a C18, 30 x 4.6 mm,
packed with 3 micron particles. Loading Mobile phase A
was 95% water and 5% acetonitrile. Loading phase B was
acetonitrile. Loading phase C was a solution containing
45% acetonitrile, 45% isopropanol, and 10% acetone.
Loading phase D was water with 0.1% ammonium
hydroxide. Eluting Mobile phase A was 90% acetonitrile
and 10% water. Eluting Mobile phase B was 90% water
and 10% acetonitrile. The appropriate gradients and flow
rates are described in Table 1.
Key Words
• Aria TLX-2
System
• TSQ Quantum
Ultra
• Clinical Research
Application
Note: 427