Bioanalytical validation for simultaneous quantification of non-aromatic steroids in follicular fluid from cattle via ESI-LC–MS/MS

• Quantitative analysis of different classes of steroids in farm animals.
• Cost-efficient quantification with no need of isotopic standards or acetonitrile.
• Double quantification of 5 steroids in 200 μl of follicular fluid.
• Internal standardization by use of dexamethasone.
• Validation of the analytical method.

The family of steroid hormones is quite attractive for the approach of phenotype monitoring in farm animals. Therefore, we developed a new protocol for the quantitative analysis of natural steroids in follicular fluid from dairy cows. The corresponding steroid profile, which consists of progesterone, corticosterone, hydrocortisone, testosterone, and androstenedione covering three distinct steroid classes, was determined by LC/MS. Quantification is achieved by use of steroid standards diluted in steroid-free follicular fluid as calibrators. Thus, the new protocol does not require deuterated standards. In order to correct for conditional performance of the analytical system we have used dexamethasone as an internal standard. The method was validated according to EMA guidelines. Within- and between-day variations were below 20% for most parameters assessed. All steroids assessed had lower limits of quantification in the range of 2.1 to 4.4 ng/ml. We have established a simple and sensitive analytical system in order to step towards a broader and cost-efficient phenotyping analysis in follicular fluid from dairy cows.