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This study determined the fat content and the concentrations of endogenous estrogens of estrone (Ε1) and estradiol (Ε2) in female fat and glandular breast tissue of three women. At the same time, the respective concentrations were studied in female plasma and plasma from female rats. More specifically, quantities of estrogens were imported in women and rat samples, homogenised, application of solid phase extraction column, derivatization for breast tissue samples and dissolving with a mixture of acetonitrile: water for plasma samples before subjected to analysis by means of GC-MS/MS.The results on human tissue samples gave satisfactory values with acceptable recoveries,for the middle and high estrogen concentrations (E1=1000fmol/g & 10000fmol/g, while E2=500fmol/g & 3000fmol/g) that were used, unlike the low concentration standards (E1=100fmol/g & E2=75fmol/g). In samples of female plasma three concentrations were used, E1=35fmol/ml & 100fmol/ml & 800fmol/ml, while E2=15fmol/ml & 125fmol/ml & 800fmol/ml) which gave equally good results with acceptable recoveries for approximately all concentrations that were used. The respective rat plasma samples, with only one concentration used (E1=E2=35fmol/ml), the experiment was performed in this case twice to find acceptable recoveries because of the unacceptable results that were found. Also examined whether the solid phase extraction column enhanced the performance of estrogen conjugates. Conjugated estrogens were used which were subjected to extraction by the above column before analyzed by means of HPLC-MS/MS method. According to the results, observed that the use of this column increases the sensitivity of analysis contributing to find accurate results. Moreover, the effectiveness of the filtration column was studied (PHREE) for removing the matrix effects in plasma samples. In this case female plasma was used twice and conjugated estrogen only once. As the plasma samples so and conjugated estrogens were extracted by filtration column (PHREE) before analysis by means of HPLC-MS/MS method. The results showed that the use of the filtration column (FREE) was not effective in removing interference that occur after the application of the plasma matrix, and when applied to conjugated estrogens observed complete loss of quantities which were used.Then, took place external calibration for quantitative analysis of estrone-glucuronide and estradiol-3-sulfate. The above estrogen conjugates were used and were dissolved with a mixture of acetonitrile: water before subjected to analysis by means of HPLC-MS/MS method. The same procedure was carried out three times. The overall results which obtained after completion of the three calibration gave acceptable linearity and variance of homogeneity only in case of estradiol-3-sulfate unlike estrone-glucuronide. Finally, taking into account only the acceptable calibration, assessed whether it is reliable to give acceptable accuracy and precision when using samples of breast tissue and plasma. The results both for accuracy and precision were found unacceptable.