Monitoring of progestins: Development of immunochemical methods for purification and detection of levonorgestrel

A polyclonal antibody (Ab) for the progestin levonorgestrel (LNG) was generated, and immunochemical assays for its detection, clean-up and concentration were developed. A highly specific microplate diagnostic assay for the detection of LNG was developed that used the enzyme linked immunosorbent assay (ELISA) method. The LNG ELISA developed was sensitive and reproducible; it exhibited I50 and I20 values of 3.3 ± 1.8 ng mL−1 and 0.6 ± 0.4 ng mL−1, respectively, and the Abs did not cross react with any of the tested steroid hormones. The above Abs were used to develop a sol–gel-based immunoaffinity purification (IAP) method for concentration and clean-up of LNG that is compatible with subsequent immunochemical or instrumental chemical analytical procedures, such as liquid chromatography followed by mass spectrometry (LC–MS/MS). Development of the columns included successful entrapment of Abs within a tetramethoxysilane (TMOS)-based SiO2 polymer network. The Abs could bind the free analyte from solution, and the bound analyte could be easily eluted from the sol–gel matrix at high recoveries. The Ab selectivity towards the antigen was high, in both ELISA and the sol–gel columns, but the entrapped Abs cross-reacted with two other steroid hormones – ethynylestradiol (EE2) and nortestosterone (NT) – which share similar epitopes with LNG, despite the lack of cross reactivity in the ELISA. The validity of the method was investigated by LC–MS/MS and a good analytical correlation was obtained.