[Establishment and validation of an LC-MS/MS-based determination method for 17-hydroxyprogesterone in canine serum]

A reliable determination of steroid hormones such as 17-hydroxyprogesterone has long been restricted by the limitations of immunological methods. Assays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or chemiluminescence immunoassay (CLIA) show significant cross-reactions, interferences and pronounced variability depending on the test, which limits their diagnostic value.

The aim of this work was the methodological establishment and validation of an LC-MS/MS-based determination method for 17-hydroxyprogesterone in order to provide a specific and robust alternative to immunological test systems.

The method was developed using stable isotope standards and included systematic testing of linearity, sensitivity, precision, recovery, and matrix effects. Subsequently, initial canine serum samples were analyzed as part of routine diagnostics to illustrate its applicability in a veterinary setting.

The method demonstrated excellent analytical performance with a detection limit of 0.044 ng/ml, high linearity (R²>0.999) and low coefficients of variation (<5%). Cross-reactions, as observed with immunological systems, were ruled out.

With the establishment of a LC-MS/MS-based 17-hydroxyprogesterone determination method, a highly specific and precise technique is available, which is in contrast to immunologically based test systems. It thus provides a reliable basis for canine endocrinologic diagnostics and steroid hormone analysis.