Traceability of Five Steroid Hormones in Butter Investigated by Gas Chromatography-combustion-isotope Ratio Mass Spectrometry
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Abstract:
A gas chromatography-combustion-isotope ratio mass spectrometry method was developed for investigating the traceability of five steroid hormones (estrone, α/β-estradiol, estriol and progesterone) in butter. The samples were first extracted by ethylacetate/cyclohexane (1:1, V/V). The sample was extracted with ethyl acetate-cyclohexane (1:1, V/V), purified by gel permeation chromatography (GPC) and semi-preparative liquid chromatography (Pre-HPLC), separated through a HP-5MS column (30 m × 0.25 mm, 0.25 μm), before traceability analysis by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and qualitative and quantitative analyses by GC/MS. The δ13C values of the exogenous hormones in butter were <-30‰ while the δ13C values of endogenous hormones were >-27‰, among which the δ13C value of exogenous progesterone in butter was -30.59‰±0.12‰ and the δ13C value of endogenous progesterone ranged from -26.83‰±0.25‰ to -23.80‰±0.33‰. One-way analysis of variance (p-value=0.009<0.05) indicated a significant difference in the δ13C values between the endogenous and exogenous progesterone. The simulation experiments showed that the δ13C value of the sample spiked with an exogenous hormone was homologous with the δ13C value of the exogenous hormone. The sensitivity of the method for the five hormones was in the range of 15~100 ng. The within-batch precision of the method ranged from 0.11‰ to 0.17‰ and the between-batch precision was 0.16‰. The step of Pre-HPLC fraction collection was critical for isotopic fractionation and analysis accuracy. The obtained results showed that the established method is accurate and specific, and GC/C/IRMS is an effective tool for investigating the origin of steroid hormones.
