LC-MS/MS method development and analysis of steroid hormones in different biological matrices

The main focus of my PhD project is the development of analytical methods that can identify the steroidogenic endocrine patterns in different biological matrices. Specifically, I investigated an LC-MS method for identifying and quantizing the main steroids hormones in different milieu. The objective was to identify the specific steroid pattern in mice to which PCOS had been induced and further treated with myo-inositol. However, given the lack of reference values for serum steroid hormones in mice, we have been obliged to previously ascertain how hormones levels could fluctuate in a normal population. Therefore, we extended the analytical measurements to healthy mice without PCOS. The data obtained will be used to establish a reference data repository, and to provide a better starting point for subsequent endocrine studies. Those studies are undergoing but preliminary results gathered so far confirm the validity of the LC-MS method. Concomitantly, we planned to ascertain the steroids output obtained directly from cultures of theca and granulosa cells. We therefore decided to include another experimental group, comprising ovarian cells from bovine. Mouse and bovine are two reference animal models to study the reproductive, endocrine system. In order to confirm the validity of LC-MS method and matrices extraction protocol I decided to test human serum of women and men of childbearing age. In fact, in this case, the concentration of steroid hormones is higher than in mice. LC-MS assessment of steroid hormones presents several criticalities, one of that is structural similarities explain that signal recognition can overlap between two similar molecules. To overcome this problem, two separate chromatographic methods were chosen, one for testosterone, androstenedione, DHEA, DHT and progesterone and one for estrone and 17β-estradiol, using the same mobile phase and polarity as the instrument but with two different chromatographic columns. The best mobile phase was found to be H2O:MeOH + 0.1% ammonium formate while the two chromatographic columns have two different stationary phases, Phnyl Bridge and C18, respectivetly for estrogens and other analytes. The two chromatographic runs have a duration of 7 minutes each and the flow gradient was as follows: MeOH 50% from 0 to 0.5 min., the up to 75% from 1 to 5 min and at the end 98% from 5.5 to 7 min for the elation of estrogens. The other flow gradient was as follows: MeOH 70% from 0 to 4 minutes, then up to 98% to 7 minutes. The flow rate is 0.3 ml/min and the oven temperature is 40°C. The extraction method's assessment is determined by the amount of sample to be extracted. A protocol has been implemented for the culture medium to concentrate the sample. We use a C18 pre columns in order to concentrate the high amount of cell culture supernatant. Due to the smaller quantity of mice and human serum, the protocol recommends liquid-liquid extraction using a mixture of hexane:ethyl acetate 9:1.