Determination of the origin of progesterone in butter by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)

The origin of progesterone in butter was investigated using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GC/C/IRMS has been rarely used to investigate the origin of natural steroid hormones, which are often found in some foods of animal origin used as feed additives. In this study, an effective pretreatment method for a butter sample was developed using gel permeation chromatography (GPC). The butter sample was dissolved with ethyl acetate-cyclohexane (1/1, v/v). After GPC pretreatment and HPLC purification, the sample was analyzed for progesterone on a GC/C/IRMS system. The delta C-13 values of endogenous progesterone in butter were found in the range from -26.77 +/- 0.26 parts per thousand to -24.78 +/- 0.32 parts per thousand and the delta C-13 value of exogenous progesterone spiked in the sample was about -30.57 +/- 0.12 parts per thousand. One-way analysis of variance (ANOVA, P-value = 0.01 < 0.05) indicated that there was a highly significant difference between the endogenous and exogenous delta C-13 values of progesterone. The sensitivity of the method for progesterone was determined to be from 10 to 100 ng. The within-batch precision of the method ranged from 0.09 to 0.15 parts per thousand and the between-batch precision was 0.11 parts per thousand. We found that the HPLC fraction collection step was a critical factor affecting the isotopic fractionation and measurement accuracy. GC/C/IRMS can be a powerful tool to investigate the origin of natural hormones.