Environmental Article

Enhanced Resolution of Endocrine Disrupting Hormones

Using an Allure® Biphenyl Column and LC-TOFMS

By Robert Freeman, Environmental Innovations Chemist, Rick Lake, Pharmaceutical Innovations Chemist, and Lydia Nolan, Innovations Chemist

Enhanced selectivity for closely related hormones.
Complete resolution of 7 common sex hormones in less than 8 minutes.
Increased confidence in identifications, using a LECO TOFMS system.

Endocrine disrupting chemicals in the environment are a topic of growing concern. Evidence suggests that the developmental and reproductive systems of both fish and wildlife have been affected.1 A variety of commonly used chemicals have endocrine disrupting properties, but the sex hormones (estrogens, progestogens and androgens) carry the most estrogenic potency.2 The primary sources are believed to be human excretion and agriculture runoff. Since these compounds generally are not affected by standard wastewater treatment practices, it is believed they are routinely discharged into receiving streams. For this reason, we sought to develop a procedure to detect endocrine disrupting hormones in aqueous matrices.

Chemically, the sex hormones 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 groups produce chemical diversity. Estrogens possess a hydroxyl group at position 3, while progestogens and androgens possess a carbonyl group (Figure 2). Typically a complex functional group at position 17 denotes a synthetically produced steroid.

Figure 1 Separations of steroids are especially challenging because all steroid molecules are based on a common structure.

Figure 2 Estrogens include a hydroxyl group at position 3, progestogens and androgens include a carbonyl group.

Estrogens
17α-estradiol	estrone	17β-estradiol
estriol	ethynyl estradiol
Progestogens
progesterone	norethindrone	norgestrel
Androgens
testosterone	19-nortestosterone	17α-methyltestosterone

Because steroids are neutral compounds, we evaluated both alkyl (i.e., C18) and phenyl stationary phases to determine the optimum phase for resolving steroid hormones. Alkyl stationary phases separate analytes on the basis of overall hydrophobicity. Phenyl phases offer a different separation mechanism: interactions among π-π electrons, between the phenyl ligand and the analytes. Often, these π-π interactions can produce alternate and enhanced selectivity.3

A downside to phenyl phases is that they typically show only moderate retention, compared to octadecylsilyl (ODS) alkyl phases. In contrast, the Allure® Biphenyl phase — a surface chemistry consisting of two phenyl groups bonded end-to-end — provides a greater concentration of phenyl groups, in sterically favorable positioning, and thereby increases π-π interactions. An Allure® Biphenyl column exhibits an overall increase in retention capacity and analyte interaction, and provides highly effective separations of compounds exhibiting differences in π-π interactions (Figure 3).

Figure 3 An Allure® Biphenyl column provides superior selectivity and retention for steroids (UV detection).

estriol
17β-estradiol
17α-estradiol
17α-ethynyl estradiol
testosterone
estrone
norethindrone

Allure® Biphenyl column

C18 Column

Columns:	150mm x 4.6mm, 3µm
Mobile phase:	water:acetonitrile, 50:50
Temperature:	ambient
Flow rate:	1.5mL/min.
Detection:	UV, 254nm

Figure 4 Sensitive analysis of steroids, using an Allure® Biphenyl column, and LECO Unique® TOFMS.

Estrogens at 5ng on column (-ESI)

Androgens at 1ng on column (+ESI)

Sample:

Inj.:

5µL

Sample diluent:

acetonitrile

Column:

Allure® Biphenyl
(cat.# 9166352)

Dimensions:

50 x 2.1mm

Particle size:

3µm

Pore size:

60Å

Conditions:

Mobile phase:

A) water + 1mM NH4OH

B) acetonitrile

Time (min.)	%B
0	45
5	70
8	70
9	45
15	45

Flow:

0.3mL/min.

Temp.:

ambient

Det.:

Leco Unique® TOFMS

To monitor steroid sex hormones in water, we first developed an extraction procedure, using styrene-divinylbenzene solid phase extraction disks and methyl tert-butyl ether (MTBE) as the extraction solvent. We conditioned the extraction disks with acetonitrile and MTBE to remove any potential interferences. After rinsing the disk with distilled water and loading the disks with one liter of sample we used 10mL of MTBE to elute the sample. Prior to analysis the final 10mL extract was concentrated to 2mL and exchanged to acetonitrile.

We recognized that the complexity of environmental sample mixtures and matrices often would make difficult a complete chromatographic separation of the steroid sex hormones by HPLC, and qualitative detection with a non-selective detector (UV-Vis). Mass spectrometry, with secondary separation based on m/z, increased our confidence in the qualitative identifications. We selected LECO Corporation’s Unique® LC-TOFMS system for its high data acquisition rate — 100 spectra/sec. The ChromaTOF® software Peak Find algorithm can deconvolute closely eluting peaks, and mass can be determined accurately, to within 5ppm, to calculate possible molecular formula. Because the ionization potential differs among the groups of steroid sex hormones, both negative and positive ESI were used. The estrogens were amenable to negative ESI, while the androgens and progestogens showed much greater sensitivity when we used positive ESI (Figure 4). We believe this difference is because of the differing functional groups at position 3.

These analyses demonstrate that the Allure® Biphenyl stationary phase, through π-π interactions, offers excellent selectivity for compounds with unsaturation differences in their hydrocarbon ring structures. Additionally, the secondary separation power of the Unique® TOFMS system and ChromaTOF® software allows overall analysis time to be reduced, through optimized column dimensions and run conditions, while qualitative identification is maintained.

References

www.epa.gov/scipoly/oscpendo/
Kuster, M., M.J. Lopez, and D. Barcelo, Estrogens and Progesterons in Wastewater, Sludge, Sediments, and Soil, pp. 3 Handbook of Environmental Chemistry
www.restek.com/fantasia/pdfCache/
580020.pdf