Comparison of pregnenolone in serum following extraction with LLE, Bond Elut C18, Bond Elut Plexa, and Bond Elut NEXUS. The method was validated for accuracy, precision, sensitivity, linearity, and through the analysis of certified plasma standard reference material. Mean concentrations were used in the calculation of the percent contribution. In human serum, the LODs ranged from 0.11 ng/mL (estrone) to 0.35 ng/mL (pregnenolone), and the LOQs were between 0.38 ng/mL (estrone) and 1.18 ng/mL (pregnenolone) (Table 3). Furthermore, 15 analytes were found in plasma SRM1950, indicating the feasibility of our method in the analysis of steroid hormones in urine and serum/plasma. The results presented in this study suggest the applicability of MEKC as a separation tool allowing the simultaneous and sensitive determination of seven steroid hormones in complex matrices such as urine samples. The elaborated MEKC method was developed to estimate the levels of seven steroid hormones in human urine samples from amateur weight-lifters using hormonal doping (13 persons), amateur weight-lifters not using hormonal doping (three persons) and healthy volunteers (five persons). The values of concentrations of steroid hormones (in ng mL−1) identified in urine samples of amateur weight-lifters, not using hormonal doping. The values of concentrations of steroid hormones (in ng mL−1) identified in urine samples of amateur weight-lifters using hormonal doping. The extraction procedures used in this study, which were based on LLE (Procedures 1 and 2), proved to not be efficient enough for the isolation of steroid hormones from urine samples. Few earlier studies employed lengthy chromatographic separation time to isolate and resolve individual analytes 5,19. LOD and LOQ of target analytes in urine were calculated as 3 and 10 times the standard deviation (SD), respectively. The inter-day CV was measured by repeated injection of fortified samples on three different days. Processed samples were stored in a refrigerator prior to analysis. The combined eluate was dried under nitrogen gas and resuspended in 120 µL of sample-reconstitution solution composed of 30% acetonitrile (v/v) in water. In 2013, the Centers for Disease Control and Prevention published a reference measurement procedure, aiming to standardize testosterone measurements . The total analysis time was 4.5 min, which was further reduced to 1.2 min per sample using a multiplex LC system. The transition used for the quantitation of unknown samples and QCs was the same as used for the calibrators. The recovery calculation for stripped serum was calculated by comparing the response of the extracted sample to that of the post-extracted spiked sample × 100% in unstripped serum with the blank also being unstripped serum (i.e., unaltered serum). In addition, unstripped serum QC2 and QC3 were prepared from Endo QC1 (50 pg/mL) by spiking solution of 5,000 pg/mL and 50,000 pg/mL. Androgens and progestogens were detected in their original forms, whereas estrogens were quantified as DC-modiXfied products (naming as E1-DC, E2-DC, and E3-DC). To diminish contamination, LC fluid for the first 1 min was discarded by using exchange value. The flow rate and column temperature were set as 0.5 ml/min and 65°C. The gradient was set as 0–0.2 min 60% B, 0.2–3.5 min 60–100% B, 3.5–4.5 min 100% B, 4.5–4.6 min 100–60% B, and 4.6–6 min 60% B. An Ekspert ultraLC 100-XL system coupled with AB SCIEX 4500 QTRAP mass spectrometer was utilized to perform the detection of sex hormones. The whole study was supervised under the Ethics Committee of Renmin Hospital of Wuhan University and abided by the Declaration of Helsinki principles. For instance, gas chromatography-mass spectrometry (GC-MS) methods provide high sensitivity, selectivity and accuracy. These methods are highly sensitive, but lack selectivity due to nonspecific antigen-antibody interactions . Steroid hormones are synthesized through a cascade-like pathway in the adrenal cortex, the gonads and the placenta and are released into the blood stream to act in both peripheral target tissues and the central nervous system. Dansylation of estrogens significantly improved their sensitivities (~11- to 23-fold) and chromatographic separation.
