Biomedical Engineering Reference
In-Depth Information
SPE. However, LLE may reduce the sample loss, experiment procedures and errors,
and save time in comparison to SPE [
8,
25,
28
]. In contrast, if a larger amount of
sample is available, e.g., 2-10 mL of urine or 1-10 L of environmental water, SPE
is a better choice, because it concentrates the sample and minimizes the interfer-
ences from other materials, leading to a higher sensitivity and selectivity of the
method or test [
34,
37,
39
]. The sample extraction throughput can be significantly
enhanced by using automated 96-well SPE plates [
31,
49
] .
2.3
Derivatization
Chemical derivatization is a standard procedure for GC-MS analysis of steroid hor-
mones, because steroid hormones are not volatile to go through GC column [
1,
6
] .
The major concerns of derivatization reagents for GC-MS analysis of steroid
hormones are the completeness of the derivatization reaction and the volatility of
hormone derivatives. The typical derivatization reagents for GC-MS samples
are silylation reagents, e.g.,
N
-methyl-
N
-tri fl uorotrimethyl acetamide (MSTFA,
[
37,
39
] ) and
N
,
O
-bis(trimethylsilyl) tri fl uoroacetamide (BSTFA, [
33
] ), which react
with both alcoholic and phenolic hydroxy groups on steroid hormone molecules.
One of the major advantages of LC-MS/MS over GC-MS or GC-MS/MS is that
steroid hormones may be analyzed directly by LC-MS/MS without derivatization
procedures, which are time-consuming and tedious [
22,
50-
53
] . However, a number
of studies demonstrated that the chemically derivatized steroid hormones were
significantly more sensitive to LC-MS/MS detection than the underivatized hor-
mones, because the neutral molecules of estrogens and metabolites might not be
effectively ionized under electrospray ionization (ESI) or atmospheric pressure
chemical ionization (APCI) modes [
4,
21,
25,
54,
55
]. In order to enhance the
steroid hormone molecules sensitivity for LC-MS/MS analysis at pg/mL level,
chemical derivatization is an effective technique for analysis of steroid hormones
and metabolites. A list of derivatization reagents and application examples for
steroid hormone analyses by LC-MS/MS and GC-MS are presented in Table
3
.
An ideal derivatization reagent is able to react with steroid hormones and metab-
olites selectively and quantitatively under mild conditions within a short time, and
those hormone derivatives are stable and easily ionized during LC-MS/MS analysis.
Based on their functional groups, the derivatization reagents used for LC-MS/MS
analyses of steroid hormones and metabolites may be classified into seven major
classes:
1. Hydrazide, e.g., (carboxymethyl)trimethylammonium chloride hydrazide (Girard
T reagent) [
4,
56
] , and
p
-toluenesulfonhydrazide [
36
] ; and hydroxylamine [
25
]
2. Benzyl bromide, e.g., penta fl uorobenzyl bromide [
55,
59
] and 4-nitrobenzyl
bromide [
60
]
3. Fluorobenzene or fl uoropyridine, e.g., 2,4-dinitro-5- fl uorobenzene analogues
[
54
] and 2- fl uoro-1-methyl-2-pyridinium
p
-toluenesulfonate [
61
]
