Biomedical Engineering Reference
In-Depth Information
deconjugation and derivatization of the steroid hormones. Thus deconjugation of
steroid hormone conjugates become a critical sample preparation procedure in
LC-MS/MS and GC-MS analyses.
Both estrogen glucuronide and sulfate conjugates may be deconjugated with
hydrochloric acid in methanol [
45
], but acid solvolysis is not a selective reaction,
and the harsh condition may cause side reactions and by-products. An alternative
deconjugation procedure applies b-glucuronidase from
Escherichia coli
to hydro-
lyze steroid glucuronide conjugates first, and then hydrolyzes steroid sulfate conju-
gates with sulfuric acid [
20
]. Steroid hormone glucuronide conjugates may be
hydrolyzed with b-glucuronidase from different sources, e.g.,
Escherichia coli
. [
38,
46
] , limpets [
47
] or
Helix pomatia
[
39
]. An attention should be paid to the fact that
b-glucuronidase from
Escherichia coli
does not affect the chemical structures of
steroid hormones during deconjugation, while b-glucuronidase from
Helix pomatia
can convert 3b-hydroxy-5-ene steroids into 3-oxo-4-ene steroids, and change
3b -hydroxy-5 a-reduced and 3b -hydroxy-5 b-reduced steroids into 3-oxo-5a -re-
duced and 3-oxo-5b-reduced steroids, respectively, because
Helix pomatia
also
contains other enzymes, including cholesterol oxidase, 3b -hydroxysteroid
oxidoreductase/3-oxosteroid-4,5-eneisomerase, and 6-hydroxylase. When
glucuronide conjugates of steroid hormones with 3-hydroxy-5-ene structure, e.g.,
progestagens and androgens, are deconjugated using b-glucuronidase from
Helix
pomatia
, the hormone quantitation may not be accurate or representative [
48
] .
However, b-glucuronidase/sulfatase from
Helix pomatia
are commonly used for
hydrolyzing both glucuronide and sulfate conjugates simultaneously, especially for
estrogens and metabolites, because the phenolic ring A of estrogens is not affected
by those enzymes in
Helix pomatia
. The deconjugation of steroid glucuronide and
sulfate conjugates is simplified as one step incubation of b -glucuronidase/sulfatase
with steroid samples at 37 °C for 20 h or at 55 °C for 3 h [
8,
28,
39
] .
2.2
Extraction of Steroid Hormones from Biological Matrices,
Environmental Water and Sediments
The techniques used for extracting steroid hormones and metabolites include pro-
tein precipitation (PP) with an organic solvent, liquid-liquid extraction (LLE), and
solid phase extraction (SPE). As shown in Fig.
2
, selection of a sample extraction
technique is based on the steroid hormone sample (unconjugated or conjugated),
quantity, matrix and the objective of an analytical method or test. For example, if a
method or test needs to analyze only the unconjugated steroid hormones in a small
volume of serum, e.g., <1 mL, a small volume of acetonitrile can precipitate the
proteins and extract steroids from the sample [
25
]. The procedure is very simple, but
the acetonitrile extract contains more nonrelated components than the extract from
LLE [
22
]. When a small volume of biological fluid is extracted by LLE with a sol-
vent, whether the sample undergoes deconjugation and/or derivatization or not, the
LLE extract is cleaner than PP extract, but may not be as clean as an extract from
