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Analysis of Hormones in Water by Automated
Solid Phase Extraction
E. Michael Thurman and Imma Ferrer, Center for Environmental Mass Spectrometry, University of Colorado, Boulder, CO, USA.

Hormones are a group of biologically active compounds
that are of concern because of their negative effects in
living organisms, such as fish and aquatic species. These
biologically-active compounds can enter surface waters
from point and non-point sources such as municipal
wastewater treatment plant (WWTP) discharges, industrial
discharges, urban runoff and animal feeding operations.
Therefore, natural and synthetic hormones are usually
detected in waters receiving wastewater discharge.
Recently, the Environmental Protection Agency (EPA)
published a Candidate Contaminant List (CCL3) which
included some of the most important emerging
contaminants in the environment. In this work, the solid-
phase extraction (SPE) for some of the hormones included
in this list was optimized.
The Horizon Technology SPE-DEX® 4790 Automated Extraction System, and the Envision® Platform Controller - SPE-DEX® 4790 Automated Extraction System - Envision® Controller Evaporation and Derivatization of Extracts:
The methanol extracts were cleaned up on Florisil columns, and evaporated to dryness under a stream of - Quatrro-micro-GC/MS/MS using Agilent 6890 GC The extracts were then derivatized to their - Column: Rxi XLB 30 m x 0.25 ID, 0.25 µm film trimethylsilyltrifluoroacetamide according to the Table 1: Purge Method
Method Summary
Collection, Preparation, and Extraction:
1. Surface water samples were taken from Boulder Creek (Boulder, CO) upstream and downstream of a WWTP. 2. A spiking mixture containing 8 labeled hormones (D Table 2: Extraction Method
medroxyprogesterone, D4-mestranol, D3-nandrolone) was used to spike water samples at a concentration of Soak Time
3. No additives were added to water samples, and no 4. The SPE-DEX 4790 system was purged using a 5. Water samples were extracted using the SPE-DEX 4790 system with the method shown in Table 2. Horizon Technology, Inc., 45 Northwestern Dr. Salem, NH, 03079 USA Analysis of Hormones in Water by Automated Solid Phase Extraction Sample Analysis:
Three surface water samples (upstream, downstream, and Helium was used as a carrier with a flow rate of 1 milliliter near a wastewater source) were analyzed with the per minute (mL/min) with the injection port maintained at methodology described in this work and several hormones 275°C. The gas chromatograph was programmed on a were successfully identified. Results are shown in Table 4. variable temperature gradient from 100°C to 310°C. For each target compound, the most abundant diagnostic ion in Conclusions
the full-scan spectrum was selected as a precursor and appropriate conditions were selected to maximize the signal The results demonstrated that the SPE-DEX 4790 using for three precursor-product transitions. The recoveries for Atlantic C18 disks can effectively extract hormone all 8 hormones were obtained by comparing the compounds from 1-L water samples in a fraction of time chromatographic areas to an external labeled standard. The (approx. 40 min). This system allows you to use the other hormones were quantified relative to the isotopic original sample bottle rinsing it with all of the extraction dilution standard (IDS) using a quantification procedure solvents before the final elution step. This rinsing step that automatically corrects for losses in the reported ensures that all the compounds are rinsed off the glass and chemical concentration based on the absolute method Results
The recoveries for the labeled hormone compounds are Horizon Technology would like to thank E. Michael presented in Table 3. In general, acceptable recoveries Thurman and Imma Ferrer of the Center for Environmental were obtained for the compounds studied. Also, the Mass Spectrometry at the University of Colorado in recoveries were very consistent in the three different water Boulder for their contributions to this Application Note. matrices studied, showing that the SPE procedure is reliable and reproducible.

Table 3: Recovery results for the labeled hormones

Recoveries (%)
Surface Water
Surface Water
Canyon Water

Table 4: Concentrations for several hormones identified in surface water samples

Recoveries (ng/L)
Surface Water
Surface Water
Canyon Water
Horizon Technology, Inc., 45 Northwestern Dr. Salem, NH, 03079 USA


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