Previously we described the construction and properties of a rapid yeast bioassay stably expressing human estrogen receptor a (hERa) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, this yeast estrogen assay was validated as a qualitative screening method for the determination of estrogenic activity in calf urine. This validation was performed, according to EC decision 2002/657, which prescribes the determination of the detection capability (CCß), the specificity/selectivity and the stability/ruggedness/applicability. To determine these performance characteristics, twenty blank urine samples of 19-week-old calves were collected and spiked with 17ß-estradiol (E2ß) at 1 ng ml-1, diethylstilbestrol (DES) at 1 ng ml-1, 17a-ethynylestradiol (EE2) at 1 ng ml-1, a-zearalanol at 30 and 50 ng ml-1 or mestranol at 10 ng ml-1. Following enzymatic deconjugation and solid phase extraction, 100 µl equivalents of these blank and spiked urine samples were screened for estrogenic activity in a 96 well plate using the yeast estrogen bioassay. All of these low estrogen spiked urine samples could be distinguished from the blank samples as all spiked samples gave a signal above the determined decision limit CCa and the mean responses of the spiked samples were higher than the determined detection capability CCß. As this CCß criterion is met, these spiked samples have a lower than 5% probability to be classified as a false negative. The specificity of the method was determined with blank urine samples spiked with a high dose of testosterone or progesterone (1000 ng ml-1). No response to these substances was detected in the yeast estrogen bioassay. There was also no interference of a high dose of testosterone or progesterone on the response of a low dose of the estrogens. Stability of urine samples was checked with spiked urine samples that were kept frozen for up to 90 days, showing that urine samples could be stored at -20 °C for up to 60 days without changing the screening result of the assay. This method has been in routine use at RIKILT for more than one year.
- tandem mass-spectrometry
- bovine urine