Biomedical Engineering Reference
In-Depth Information
The first class of derivatization reagents, hydrazide reagents and hydroxylamine,
react with most of the ketolic steroid hormones and metabolites (ketone group on
C
3
, C
17
, or C
20
), i.e., androgens, progestagens, corticoids, and ketolic estrogens
[
4,
25,
36,
56
]. However, they are not suitable for steroid hormones without ketolic
group(s), such as estradiol, estriol and their related metabolites. Hydroxylamine is a
typical derivatization reagent for steroid hormone profiling, because it can react
with all the ketolic hormones, and provide unique mass fragments for each steroid
moiety during LC-MS/MS analysis. When these unique mass fragments or daughter
ions are used for multiple reaction monitoring (MRM) quantitation, the method
selectivity is much higher than those using a daughter ion from a derivatization
reagent, e.g.,
m/z
171 for dansyl ion [
25
] .
The second to seventh classes of reagents react with hydroxyl steroid hormones
and metabolites, especially estrogens. Pentafluorobenzyl bromide estrogen deriva-
tives, belonging to the second class, are sensitive to both ESI
+
[
1
] and APCI
−
[
55,
61
]
modes, and usually have lower limits of quantitation (LOQ) values under APCI
−
mode than those of derivatives of dansyl chloride and 2-fluoro-1-methyl-pyridinium
p
-toluenesulfonate under ESI
+
mode, because there were little interference from
analogue compounds and the matrix background under APCI
−
mode. Nevertheless,
the derivatization reaction of estrogens with pentafluorobenzyl bromide was ten
times longer than the derivatization reaction with dansyl chloride (30 min vs. 3 min
at 60 °C) [
61
]. A study by Higashi et al. indicated that the derivatization reac-
tions of estrogens with 4-nitrobenzyl bromide (the second class), 2,4-dinitro-
fluorobenzene (the third class) and 4-nitrobenzoyl chloride (the sixth class) were
not as complete as the reaction with 4-nitrobenzene sulfonyl chloride (the fourth
class) [
60
] .
The fifth class, carboxylic acid
N
-hydroxysuccinimide ester, is also not as reac-
tive as sulfonyl chloride, and its derivative is not as sensitive to LC-MS/MS either
[
21
]. The sixth class of reagents, carbonyl chloride and carboxylic acid anhydride,
can react with both phenolic and alcoholic hydroxyl groups of steroids. However,
the selectivity, speed, and completeness of derivatization reactions of these reagents
with steroids are not as good as those of sulfonyl chloride derivatization reagents
[
60,
64,
66
]. Since the other four classes of derivatization reagents, sulfonyl
chloride, benzyl bromide, carboxylic acid
N
-hydroxysuccinimide ester, and
fluorobenzene, are able to selectively react with phenolic hydroxyl group of estro-
gens and metabolites, the carbonyl chloride and carboxylic acid anhydride reagents
become less preferable for derivatizing estrogens and metabolites. The seventh
class,
o
-phenylenediamine, is a specific derivatization reagent for estrogen
o
-
quinones, potential carcinogens, such as estrone-2,3-quinone, estrone-3,4-quinone,
estradiol-2,3-quinone, estradiol-3,4-quinone [
67
] .
Works published so far suggest that sulfonyl chloride is a preferred reagent for
derivatizing estrogens and their metabolites, due to its reaction completeness and
selectivity. In addition, a sulfonyl chloride reagent containing a basic or preionized
nitrogen atom, e.g., on dansyl, pyridine, imidazole, pyrazole, or piperizine ring,
could significantly enhance the ionization of estrogen derivatives under ESI
+
mode,
and increase the detection sensitivity [
4,
54,
62
]. Dansyl chloride is a typical sulfonyl
