Quantitative analysis of conjugated and free estrogens in swine manure: Solutions to overcome analytical problems due to matrix effects

Although animal manure is an important source for environmental estrogens, quantitative analysis of estrogens in manure is complicated due to matrix interference. In the present study, chromatographic methods have been developed for quantification of conjugated and free estrogens in manure samples collected from pig farms. The whole manure samples, immediately after collection, were stored at 4 °C, acidified (pH ≈ 2.0) and spiked with (i) 13C-labeled internal standards to account for possible storage related degradation and (ii) deuterium labeled internal standards for calibration and quantitative analysis. The liquid samples were extracted with ethyl acetate for separating conjugated and free estrogens. The solid samples were eluted with water for desorbing conjugated hormones followed by methanol for desorbing free hormones. The water and extracts were further purified using hydrophilic–lipophilic balance and/or aminopropyl cartridges. The conjugated estrogens were analyzed using high-performance liquid chromatograph–mass spectrometer, while the free estrogens were analyzed using gas chromatograph–mass spectrometer. The extraction and calibration methods used in the present study yielded excellent sensitivity, reproducibility and >85% recovery of both free and conjugated estrogens that was independent of the manure matrix. In general, the total estrogen loads in liquid and solid samples were 5.1 mg/l and 4.93 mg/kg, respectively. This may represent the hormonal load of approximately 2.3 tons estrogen per day