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.
The extraction of testosterone and 11ß-MNT in serum (sample volume 150 μL) entailed the addition of 10 μl of stable isotopically labeled internal standard (SIL-IS) — 30 ng/mL of testosterone-2,3,4-13C3 and 11ß-MNT-d6 each in acetonitrile. One volume of spiking solutions was added to 99 volumes of charcoal-stripped cynomolgus monkey serum to obtain nominal concentrations of standard solutions. Nevertheless, using LC-MS/MS techniques to separate and measure steroid isomers like 11β-methyl-19-nortestosterone (11ß-MNT) and testosterone (Figure 1) in blood serum samples can be challenging. Finally, the proposed method was subjected to analytical validation for determination of testosterone in plasma samples. The calibration curves were prepared in stripped serum diluted with ultrapure water at equivalent volumes (Diluted Stripped Serum) while the QC samples were prepared in both stripped and unstripped serum (all unstripped serum refers to unstripped postmenopausal or premenopausal human serum). To compare specificity, the ion ratio transition of 289 → 97 to 289 → 109 was monitored for all QCs and unknown samples; no interference in the testosterone peak was observed for any tested sample prepared by either the one-step or two-step procedure. It has been also demonstrated that micro TLC equipment can be applied as a fast retention screening device as well as simple and robust quantitative tool for determination of testosterone residue containing testosterone derivatives in complex samples.
Among the 19 steroid hormones, 15 analytes were quantified at concentrations ranging from 0.14 (estrone) and 81.4 ng/mL (cortisol). Method validation parameters for the analysis of 19 steroid hormones in human serum. Method validation parameters for the analysis of 19 steroid hormones in human urine. Thus, deconjugation is an essential step to measure the total concentrations of steroid hormones.
To address this limitation, we developed a total testosterone LC-MS/MS assay on three instruments. High-throughput immunoassays often lack accuracy in lower concentration ranges (below 100 ng/dL), particularly when used for females or children. The circulating levels of total testosterone vary from 1 to 1480 ng/dL. The mass spectrometric principle is applicable to the LC/LC-MS/MS where SIL-ISs cancelled the matrix effect in both types of serum. In principle, an internal standard can cancel the matrix effect of an analyte in LC-MS analysis 38–40.
Furthermore, in this group only in one case was the presence of 17α–methyltestosterone (which is classified as an exogenous steroid) observed. The level of testosterone (T) excreted in the urine under physiological conditions is generally in the range of 20–150 ng mL−1, and in people who practice various sports, especially power sports, the level may exceed 150 ng mL−1. The advantage of the determination of this hormone in urine is the non-invasive sampling. The doping control test is the most appropriate indication of the ratio of testosterone glucuronide to epitestostrone glucuronide in urine. However, this treatment is not effective, since the anti-doping analysis in the study included the simultaneous quantification of both hormones. According to the guidelines given by Peters et al. full validation of the method must include the stability of the analyte in the sample matrix and for this tests should be established. Although many authors consider that the determination of the recovery is not as important as the designation of LOD, precision and accuracy , in this study, the recoveries for all analytes were estimated.