Environmental Analysis of Selected Estrogens and androgens: Applying Ultra-Performance Liquid Chromatography and Combating Matrix Interference

• Main Author: Rice, Stacie L.
• Format: Others
• Language: English
• Published: W&M ScholarWorks 2009
• Subjects: Analytical Chemistry; Environmental Health and Protection
• Online Access: https://scholarworks.wm.edu/etd/1539617882; https://scholarworks.wm.edu/cgi/viewcontent.cgi?article=3012&context=etd

Investigations of environmental hormone contamination commonly utilize solidphase extraction (SPE) followed by high-performance liquid chromatography / (electrospray ionization) tandem mass spectrometry (HPLC/(ESI)MS2) in the detection of estrogens. Matrix interference is widely reported. In this study, androgens were targeted alongside estrogens as environmentally co-introduced endocrine disrupting chemicals. Analytical methods were developed in parallel for detection of several hormones from each class, with comparison of protocols and instrumental parameters. Ultraperformance LC (UPLC®), an emerging technology advertised for reduced retention times, was used in place of HPLC for hormone separation. Applicability to diverse aqueous samples was tested. Matrix interference was combated with two rarely used techniques: atmospheric pressure chemical ionization (APCI) MS2 and extract purification by size exclusion chromatography (SEC). Infusion and spiking tests were performed in (ESI)MS2 and (APCI)MS2, demonstrating relative matrix disruption of hormone signals. An SEC fractionation protocol was developed and applied to extracted wastewater samples prior to UPLC/MS2 analysis. Hormone recoveries were compared to those obtained without SEC purification. In accordance with these experiments, it was found that estrogen and androgen contaminants can be simultaneously extracted using C18 SPE. This approach reduces the amount of sample, supplies, and time required in preparation for instrumental analysis. In UPLC separation, a broader gradient, slower flow rate, and increased run time were used for the androgens to counteract structural similarity and reduced polarity. The androgens were most readily detected using positive (ESI)MS2, versus negative for the estrogens, with modifier addition for signal enhancement. Electron delocalization in the estrogen and testosterone molecular structures facilitated ionization, permitting MS2 detection at or below 23 pg on-column versus 500 pg for the androgens lacking bond conjugation. Recoveries of all analytes from deionized water were 67-112% using UPLC/(ESI)MS2. Use of UPLC reduced retention times and solvent usage in comparison to HPLC, permitting adequate resolution of the hormones within 7 min in the presence of clean solvents. In environmental samples, the rapid analyses proved susceptible to matrix interference, with lack of signal resolution amidst unresolved complex mixtures. The application of (APCI)MS2 to complex samples showed promise in combating matrix interference, permitting detection of hormones spiked into wastewater that were not observed using (ESI)MS2. The ionization methods tended toward opposite matrix effects, with 140-410% recoveries (i.e. ion enhancement) from effluents using APCI and 5-120% (i.e. ion suppression) using ESI. Application of SEC prior to instrumental analysis removed some interfering compounds, allowing recoveries of 48-98% for several hormones using UPLC/(APCI)MS2.