A lower voltage caused the separation time to be longer and the phenomenon of "tailing peaks" appeared. The most optimal separation conditions prevailed when a temperature of 22 °C was employed, therefore all separations were carried out at 22 °C. The temperature has an influence on the density and the mobility of ions, as well as on the volume of the injected sample. On the other hand, too short an injection time can result in a too small amount of the sample injected into the capillary, thereby reducing the peak height or creating the absence of signals.
It is worthwhile noting that the endo QC values from the two procedures are identical, indicating that the one-step procedure offers the same assay quality. By comparison, back-calculation used only the curve prepared using the two-step procedure while the curve prepared by the one-step procedure was used as QC. 1, the signal-to-noise of the LLOQ from the one-step procedure is slightly higher than that for the two-step procedure. Data in Table 2 indicate that there is no difference for testo extracted by either procedure, indicating that neither procedure introduces detectable interference into the testo peak.
The difference of back calculated QCs at each level was within the range of 99%–102% between the two procedures (Table 2), while the difference of each back-calculated unknown was within the range of 95%–105% for the two procedures for adult sera (Table 3A). Hence, any variation from different runs, different time intervals and different working IS was eliminated. If there are no interferences in either of the two MRM transitions, the ratios obtained from standards of the calibration compared to any unknown sample should be within the acceptance criteria range, which is usually 15% . This is a powerful method to validate the specificity of measurement for endogenous compounds. Analyst 1.6.2 (Sciex, Concord, Canada) was used for data processing and analysis. A Poroshell EC–120 C18 column (50 × 3.0 mm, 2.7 µm) (Agilent Technologies, Mississauga, ON, Canada) was chosen for separation. Following 5 min of centrifugation (2000g), the organic layer was transferred for drying and the dried residue was reconstituted using 100 μL of 30% methanol.
In the group of sports enthusiasts using illegal performance enhancers, in which the average level of 17α-MT was 165.0 ± 45.7 ng mL−1, the presence of methyltestosterone can be explained by consumption or by naturally occurring testosterone metabolism . Long-term secretion of large amounts of cortisol by the human body in response to chronic stress may lead to Cushing’s syndrome. Methyltestosterone was also used in doping, as an anabolic agent and to enhancd the ability to fight in sports and even aggression, making it especially popular among people practicing martial arts, due to the increased aggression and the anabolic effects . Epitestosterone was used as a doping agent to mask the excessive use of synthetic testosterone (which should not exceed the limit of the T/ET ratio). For the purposes of doping, derivatives of testosterone and dihydrotestosterone are used to increase muscle mass, increase muscle strength and improve overall performance. Testosterone, because of its anabolic effects, is one of the basic and most used anabolic steroids in doping. After comparing the results, one month after storage recovery values were in the range 97.4%–100.2%, the average value was 98.2%, and after 3 months, the determined values were within 93.1%–100.9% and the average value was 95.3%.
350 mg (equivalent of 40 mg Testosterone Undecanoate) of test sample was taken in a 100 ml volumetric flask. Testosterone Undecanoate stock solution were used to prepare calibration standard solution in daily basis. 2 ml of stock solution were transferred in to 20 ml volumetric flask and volume to the mark with diluent. 20 mg of testosterone undecanoate chemical standard was transferred in 50 ml volumetric flask dissolved it with methanol with proper sonication. The mobile phase was only 100% methanol which was filtered 0.45 μm nylon filter and degassed in ultrasonic bath before use. Where C is the concentration in ppm of the Testosterone Undecanoate. HPLC ready deionized 18Milli-Q water was obtained, in-house, from a Milli-Q Gradient A-10 water purification system, Millipore, (Bedford, MA, USA).
Testosterone and 11ß-MNT were extracted and purified from serum via the liquid-liquid extraction method described in reference to maximize recovery of analytes and minimize the matrix effect from samples during detection . Liquid-liquid extraction is the preferred methodology for cleaning up biological samples, while also being widely used for the specific extraction of less-polar compounds , including most pharmaceutical compounds and many endogenous molecules, such as steroid hormones , , . In this study, a one-step method for liquid-liquid extraction has been compared against a two-step procedure for testosterone assays in terms of accuracy, specificity, recovery, lipid removal and baseline noise, using QCs and unknown samples. Steroid hormone concentrations measured in twenty human urine samples from the general population in New York, USA, in 2022. The method was applied in the analysis of 19 hormones in real human urine and serum/plasma.
Schematic diagram of LLE-based and CIPS-based method for sex hormone analysis in serum. The obtained upper phase (50 μl) was pipetted into water (50 μl) for LC-MS/MS analysis. The upper phase (100 μl) was collected and mixed with sodium bicarbonate solution (100 μl, 0.1 mol/l in water) and DC solution (10 μl, 10 mg/ml in ACN). And CIPS was performed by storing samples at −30°C for 10 min. Serum samples (100 μl) were combined with IS working solution (10 μl) and ACN (200 μl).

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