About
One of the most important parameters for the optimization procedure was the selection of a suitable running buffer for the effective separation of all components. Even better results were obtained using hydrophilic-lipophilic balance (HLB) cartridges, which are commonly applied for the extraction of a variety of analytes from water. This type of extraction during the analysis of steroids has been employed by many researchers 14,15,16,17,18.Testosterone, administered through intramuscular injection, skin patch and wax pellets that are inserted into a deep subcutaneous for androgen supplement therapy has been used for the treatment for hypogonadism or andropause 15-19. 1 to 2% of it is not bound with protein and represents the free state and considered the biologically active testosterone and available for tissue uptake . Testosterone is mainly is bound with albumin protein with low affinity and to sex hormone binding globulin (40-50%) with high affinity. The new method can offer comparable detecting performance as traditional LLE/SPE methods whereas exhibits obviously higher throughput. In conclusion, the present study developed a novel LC-MS/MS method for sex hormone quantification.
Steroid hormone concentrations measured in the pooled human serum purchased from Sigma-Aldrich. Representative chromatograms of all analytes in serum samples are shown in Figure S4. The method was applied in the analysis of human plasma by measuring all analytes in SRM1950. Concentrations (a) and relative distribution (b) of 19 steroid hormones measured in urine from 11 males and 9 females from New York, United States. Representative chromatograms of all analytes in urine samples are shown in Figure S3. Sixteen analytes were found with detection frequencies (DFs) ≥ 80% in urine samples.
For acid and alkali hydrolysis samples were treated with 10 ml of 1 M HCl and 1 M NaOH and then sonicate for 30 min and then stayed for 1.5 hours. During performing robustness test standard stock solution at concentration of 40 ppm Testosterone Undecanoate was used and it was found that all the criteria for system suitability was satisfactory. System suitability was determined by injecting five replicate standard solution from same vial before analyze test sample each day. It was prepared by using 5 concentrations with three replicates by diluting stock solution to the concentrations of 20, 32, 40, 48 and 60 ppm. Many analytical methods has been proposed for quantitative determination of testosterone undecanoate such as UV method 24,25, gas chromatography-mass spectroscopy 21,26,27, LCMS/ MS method 28-31, nuclear magnetic resonance , LC-QTOF/ MS .
While within 5 min, the phase separation can be well preserved. The method accuracies (as apparent recoveries) and imprecisions (as CV) for detecting sex hormones at four concentration levels After LC-MS/MS analysis, the consistency of all the targets was 87.1–108.8% from the enrolled six samples. While after storage for 10 min or longer, the volume of upper phase was stable at 110 μl. This could be ascribed to the fact that the distinction of ACN percentage in upper phase (88%) to lower phase (35%) after CIPS was relatively independent to the ACN-water proportion in initial solution.
Top Product ions from protonated 11ß-MNT; Bottom Product ions from protonated testosterone. Both compounds share the same product ions from CID, and these can interfere with each other in LC-MS/MS analysis if both have the same retention time (tR). Mass spectra of fragments from protonated 11ß-MNT and testosterone ions by collision-induced dissociation (CID). Cone voltages and collision energies were optimized to measure the analytes and internal standards by LC/LC-MS/MS. We used a Waters Xevo TQ-S MS system equipped with an electrospray ionization (ESI) source and detected 11ß-MNT and testosterone by MRM operated in the positive ion mode.
Due to the very low pH of the electrophoretic buffer the authors used reversed polarity electrodes, so the analysis time was significantly reduced. Cho et al. assessed HLB cartridges containing a copolymer sorbent that has hydrophilic and lipophilic groups and provided high recoveries for 21 endogenous corticosteroids. This is caused by the comparatively higher water solubility of the latter compared to the parent molecules.
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).
As the components of this layer are ACN and water, it can be transferred for LC-MS/MS analysis directly. Along with phase separation, targets are enriched into the upper layer owing to their hydrophobicity. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability.