A Fast and Sensitive Method for the Detection of Leuprolide Acetate: A High-Throughput Approach for the In Vitro Evaluation of Liquid Crystal Formulations

The suitability of using fluorescence spectroscopy to rapidly assay drug release by quantifying the time-dependent increase in total intrinsic protein fluorescence was assessed. Leuprolide acetate, a synthetic nonapeptide analogue of gonadotropin-releasing hormone (GnRH or LHRH), is the active pharmaceutical ingredient used to treat a wide range of sex hormone-related disorders, including advanced prostatic cancer, endometriosis, and precocious puberty. During the in vitro evaluation of drug delivery technologies for leuprolide acetate, one of the most time-consuming steps is the detection and accurate quantification of leuprolide release from formulation candidates. Thus far, the dominant means for leuprolide detection involves conventional multistep high-performance liquid chromatography (HPLC) methods, requiring sampling, dilutions, sample filtration, and chromatography, which can take up to 40 min for each sample. With the increasing demand for assay adaptation to high-throughput format, here we sought to exploit fluorescence spectroscopy as a tool to develop a novel method to rapidly assay the in vitro release of leuprolide acetate. By utilizing the intrinsic fluorescence of the tryptophan (Trp) and tyrosine (Tyr) amino acid residues present in the leuprolide nonapeptide, the in vitro release from liquid crystal formulations was accurately quantified as a function of fluorescence intensity. Here, we demonstrate that assaying leuprolide release using intrinsic protein fluorescence in a 96-well format requiring volumes as low as 100 mu L is a cost-effective, rapid, and highly sensitive alternative to conventional HPLC methods. Furthermore, the high signal-to-noise ratios and robust Z'-factors of >0.8 indicate high sensitivity, precision, and feasibility for miniaturization, high throughput format adaptation, and automation.