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Environmental
Enhanced Resolution of Endocrine Disrupting Hormones
Using an Allure" Biphenyl Column and LC-TOFMS
ByRobert Freeman, Environmental Innovations Chemist, Rick Lake, Pharmaceutical Innovations Chemist, and Lydia Nolan, Innovations Chemist
• Enhanced selectivi ty for closely related hormones.
• Complete resoluti on of 7 common sex hormones in less than 8 minutes.
• Increased confidence in identificat ions, using a LECO TOFMS system.
Endocrine disrupting chemicals in the environ­
ment are a topic of growing concern. Evidence
suggests that the developmental and reproductive
systems of both fish and wildlife have been affect­
ed.' A variety of commonly used chemicals have
endocrine disrup ting proper ties, but the sex hor­
mones (estrogens, progestogens and androgens)
carry the most estrogenic potency.' The pr imary
sources are believed to be human excretion and
agriculture runoff. Since these compo unds gener­
ally are not affected by standard wastewater treat­
ment practices, it is believed they are routinel y dis­
charged into receiving streams. For this reason, we
sought to develop a procedure to detect endocrine
disrupting hormones in aqueous matrices.
Chemically, the sex hormo nes are steroids.
Steroids are a unique class of compounds, in that
all structural variation is centered on a common
conjugated ring system (Figure 1), from which
double bonding and various functional group s
produce chemical diversity. Estrogens possess a
hydroxyl group at position 3, while progestogens
and androge ns possess a carbonyl group (Figure
2). Typically a comp lex functional group at posi­
tion 17 denotes a synthetically produced steroid.
Because stero ids are neutral compounds, we eval­
uated both alkyl (i.e., C18) and phenyl statio nary
phases to determine the opt imum phase for
resolving steroid hormones. Alkyl stationa ry phas­
es separate analytes on th e basis of overall
hydrophobicity. Phenyl phases offer a different
separation mechanism: inter actions amo ng IT-IT
electrons, between the phenyl ligand and the ana­
Iytes. often, these IT-IT interactions can prod uce
alternate and enhanced selectivity.'
A downside to phenyl phases is that they typically
show only mod erate retention , compared to
octadecylsilyl (ODS) alkyl phases. In contrast, the
Allure@ Biphenyl phase - a surface chemistry con­
sisting of two phenyl group s bond ed end-to-end ­
provides a greater concentration of phenyl groups,
in sterically favorable positioning, and thereby
increases IT-IT interactions. An Allure® Biphenyl
column exhibits an overall increase in retention
capacity and analyte interaction, and provides
highly effective separations of compounds exhibit­
ing differences in IT-IT interactions (Figure 3).
2006.03
Figure 1 Separations of steroids are especially challenging
because all steroid molecules are based on a common structure.
Cyclopentane
16
12
Phenanthrene 11 ~
Figure 2 Estrogens include a hydroxyl group at position 3,
progestogens and androgens include a carbonyl group.
Estrogens
17a- estradiol estrone 17p-estradiol
en1 ! ! - I
"" I ' , o ,
""~_0 ""~"' ""estriol
ethynylestrad iol
I I- i " ~.0 ~~
I I i
""~ ""- ~
Progestogens
progeste rone noreth indrone norgestrel
i 0#" ; i
0 o "'"
Androgens
testosterone 19-nortestoste rone 17a -methyltestosterone
" i :"
a a , , • • £t8+5 ~ ~
o "'" o "'"
Acknowledgement
Wearegrateful to Paul Kennedy, Ph.D, LECDCorporation, 3000 Lakeview Avenue, SI. Joseph, MI 49085-2396,
for his assistance with this analysis.
800-356-1688 •
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