The benefits of SLE in the development of a GLP protocol for the measurement of 17β-estradiol and testosterone in the H295R steroidogenesis assay, Test No 456

Regulators are concerned about the potential for environmental chemicals such as agrochemicals and their metabolites to perturb hormone systems. This has led to recommendations for the testing of potential endocrine disrupting chemicals. 

The Steroidogenesis H295R assay is an in vitro cell model used to investigate compound effects on steroid hormone biosynthesis, specifically 17β-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line expresses genes that encode for all the key enzymes for steroidogenesis and thus forms one of the required OECD in vitro tests (TG456) for the assessment of potential endocrine disrupting chemicals. 

Regulators are concerned about the potential for environmental chemicals such as agrochemicals and their metabolites to perturb hormone systems. This has led to recommendations for the testing of potential endocrine disrupting chemicals. 

The Steroidogenesis H295R assay is an in vitro cell model used to investigate compound effects on steroid hormone biosynthesis, specifically 17β-estradiol (E2) and testosterone (T). The human H295R adreno-carcinoma cell line expresses genes that encode for all the key enzymes for steroidogenesis and thus forms one of the required OECD in vitro tests (TG456) for the assessment of potential endocrine disrupting chemicals. 

Although it is possible to assess hormone levels with ELISA we elected to perform the TG456 assay with LC-MS/MS hormone detection, avoiding the test item interference issues reported for immunoassay-based readouts. 

We describe herein our studies on the benefits of supported liquid extraction (SLE) versus liquid-liquid extraction (LLE) and protein precipitation in the development of a robust GLP bioanalytical method for the detection of testosterone and 17β-estradiol in the steroidogenesis assay, to LLOQ levels of 10 pg/mL for each hormone.

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