Biomedical Engineering Reference
In-Depth Information
2
Sample Preparation
The major sample preparation procedures of steroid hormone analyses include
extraction, deconjugation, and derivatization, as shown in Fig.
2
. A number of
examples of steroid hormone sample preparation are summarized in Table
2
.
Application of stable isotope labeled (e.g., deuterated or
13
carbon labeled) ste-
roid hormones as the internal standard or isotope dilution is a standard of prac-
tice during quantitative steroid hormone analyses by LC-MS/MS and GC-MS
whenever possible. These isotope labeled internal standards are added into the
samples before deconjugation or extraction procedures. They undergo the same
deconjugation and/or extraction procedures and LC-MS/MS or GC-MS analysis
as the steroid samples do. The efficiency of deconjugation and/or extraction,
assay accuracy and precision are calibrated and calculated with the isotope
internal standards, leading to more accurate, precise, and robust methods and
results.
2.1
Deconjugation of Steroid Hormones
A large percentage of steroid hormones exist as glucuronide, sulfate, and glutathi-
one conjugates in body fluids and tissues. These steroid conjugates may be ana-
lyzed directly by LC-MS/MS using either electrospray ionization (ESI) [
5,
40-
43
]
or atmospheric pressure chemical ionization (APCI) [
44
] mode. However, in many
cases, the presence of glucuronide and sulfate conjugates in samples may reduce
MS detection sensitivity and the total amount of a hormone (both unconjugated
and conjugated) that can be determined by LC-MS/MS [
8
] or GC-MS [
6
] after the
Steroids in
matrics
Solvent
precipitation
Liquid/liquid
extraction
Solid phase
extraction
Hydrolyze
conjugate
Liquid/liquid
extraction
Solid phase
extraction
Derivatization
LC/MS
LC/MS
LC/MS
Derivatization
LC/MS
GC/MS
LC/MS
GC/MS
Derivatization
Derivatization
LC/MS
GC/MS
LC/MS
GC/MS
Fig. 2
Steroid hormone sample preparation procedures: extraction, deconjugation, and derivatization
