Biomedical Engineering Reference
In-Depth Information
chloride reagent used for derivatizing estrogens and metabolites from varieties of
matrices, such as river water [
50,
61
], mouse plasma and brain [
27
] , human urine
[
28
] , breast tissue [
26
] , and serum [
8
]. However, dansyl derivatives have a disadvan-
tage that the common dansyl fragment
m/z
171 from background of the derivatiza-
tion reagent and from those derivatives may have negative impacts on the method
selectivity and accuracy during LC-MS/MS analysis, especially for those isomers
with the same molecular ions and fragments, because the elevated dansyl fragment
background noise may reduce the analyte signal-noise ratio, and cross interfere
quantitation of other analytes with the same fragments [
8,
25,
28
] .
3
Comparison of LC-MS/MS, GC-MS, and Immunoassays
3.1
LC-MS/MS and UHPLC-MS/MS vs. GC-MS
and GC-MS/MS
The bioanalytical methods developed in recent years focused more on LC-MS/MS
and GC-MS/MS techniques, because the earlier studies demonstrated that LC-MS/
MS and GC-MS/MS are significantly more sensitive in analyzing estrogens and
metabolites than LC-MS and GC-MS [
18,
68
]. Analyzing both unconjugated and
conjugated steroid hormones directly is the major advantage of LC-MS and
LC-MS/MS over GC-MS and GC-MS/MS, because the sample preparation proce-
dures of deconjugation and derivatization can be avoided [
5,
7,
40-
42,
44
] . In
complex samples, separation of steroid hormones and metabolites by LC or GC is
still one of the major concerns, because many steroid hormones and metabolites
have the same molecular weights and MS fragments, and they may interfere with
each other if not separated. For example, 2-hydroxyestrone and 4-hydroxyestrone
have same molecular weight, even after they are derivatized with MSTFA for
GC-MS analysis [
48
] or with dansyl chloride or
p
-toluenesulfonhydrazide for
LC-MS/MS analysis [
8,
35
]. If the derivatives are not separated, GC-MS or
LC-MS/MS is unable to distinguish the derivatives of 2-hydroxyestrone from those
of 4-hydroxyestrone, whether the silylated, dansylated, or hydrozone steroid
fragments are used for quantitation.
An LC column with a length of 150 mm can separate up to 23 steroid hormones
and metabolites [
20
]. A typical LC-MS/MS method developed by Xu et al. was able
to separate 15 estrogens and metabolites using a 150 × 2 mm, 4 mm LC column,
which had a run time of 100 min [
8,
28
]. The separation efficiency can be improved
by using smaller particle size LC columns, e.g., 100 × 2.0 mm, 2.5 m m column [
69
]
or 50 × 2.1 mm, 1.8 m m column [
21
], also leading to a significantly reduced run time
(e.g., less than 30 min). Similarly, ultra high performance liquid chromatography
(UHPLC) is able to significantly improve separation efficiency and to reduce run
time [
40,
70-
72
]. Two-dimensional (2D) LC-MS/MS with column-switching tech-
nique has been used for determination of unconjugated and conjugated estrogens in
