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Monitoring of estradiol and its metabolites in biological samples is essential for the accurate diagnosis of a number of endocrine diseases. In this study, a sensitive, precise and specific GC-MS/MS method for the quantification of 17β-estradiol (17-BE) and its main metabolite, 2-methoxyestradiol (2-MEOE), in plasma was developed and validated. Plasma concentrations of these steroids are currently investigated as diagnostic markers for pre-eclampsia, a systematic disorder of pregnancy and a leading cause of maternal and fetal morbidity and mortality worldwide. The method comprised treatment of the plasma sample by protein precipitation and subsequent isolation of steroids by solid phase extraction, derivatization of steroids by trifluoroacetic anhydride and GC-MS/MS analysis of the derivatized steroids. The large volume (10 μL) injection with the assistance of a Programmed Temperature Vaporization (PTV) injector in solvent split mode allowed a substantial increase in the sensitivity of the method. The ion trap MS was operated in optimized Product Ion Scan. By increasing the damping gas flow in the ion trap from the conventional 0.3 mL min(-1) to 2 mL min(-1), ion fragmentation was reduced and the instrument response was enhanced substantially. As a result, mass spectra with predominant molecular ions were acquired and molecular ions of the steroids of interest were used as precursor ions thus increasing specificity of the method. Under optimized GC-MS/MS conditions in product ion mode, the Limit of Detection (LOD) of the analyzed steroids ranged from 18.4 pg mL(-1) for 17-BE to 5.5 pg mL(-1) for 2-MEOE (S/N=3). The instrument response was linear in the investigated concentration range from 0.1 to 10 ng mL(-1) with R(2)>0.99 for 17-BE and 2-MEOE. The intra-batch accuracy obtained for quality control samples at the concentration levels of 0.1, 1, 3, 7 ng mL(-1) ranged from 94.9% to 109.9% for 17-BE and from 99.9% to 104.5% for 2-MEOE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Risk factors for atherosclerosis such as hypertension, type 2 diabetes, obesity and dyslipidemia affect endothelial function and stimulate adhesion molecules expression. The aim of the study was to examine endothelial activation in type 2 diabetes and hypertension as indicated by adhesion molecule levels and further to investigate whether the coexistence of the above conditions has a different effect., Methods: Serum levels of soluble E-selectin, ICAM-1 and VCAM-1 were measured in 17 hypertensive type 2 diabetic patients (DM-HY), 32 normotensive type 2 diabetic patients (DM), 11 hypertensive nondiabetic patients (HY) and 15 healthy subjects., Results: In diabetic patients (either DM-HY or DM), soluble E-selectin levels were significantly increased compared to healthy subjects (p<0.001). In HY patients, both sE-selectin (66.44+/-71.59 vs. 29.42+/-15.56 ng/ml, p=0.033) and sVCAM-1 (1529+/-433.33 vs. 1027+/-243.56 ng/ml, p=0.03) levels were found significantly higher compared to healthy subjects (p<0.05). The coexistence of diabetes and hypertension (DM-HY) did not have an additive effect on circulating adhesion molecules levels compared with the levels observed in either diabetes or hypertension. Systolic and diastolic blood pressure (BP) were independent factors correlated respectively with sE-selectin and sVCAM-1 levels (R=0.454, p=0.034 and R=0.578, p=0.005) in nondiabetic subjects (hypertensive and normotensive)., Conclusions: Type 2 diabetes mellitus and hypertension induce endothelial activation as indicated by elevated levels of soluble adhesion molecules. This effect is not different when comorbidity is present.