The chemical structures of testosterone and 11ß-MNT are very similar and cannot be quickly separated by reverse-phase chromatography. Although each fragment has different relative abundance in the CID spectra of 11ß-MNT and testosterone, cross interference is inevitable when 11ß-MNT and testosterone are simultaneously measured by MS/MS. The MRM transitions of analytes and internal standard are testosterone (289→97), 11ß-MNT (289→109), testosterone-2,3,4-13C3 (292→100) and 11ß-MNT-d6 (295→277), respectively. The mobile phases compositions remained the same for 12 minutes, and the columns were re-equilibrated for 3 minutes. On the other hand, metabolites of most steroid hormones produced in the body are excreted with urine, which allows the assessment of full hormonal profiles. The determination of steroid hormone levels in blood samples may be impaired due to the oscillatory secretion of these compounds according to the circadian rhythm and the menstrual cycle. The most widely used biological materials for the investigation of these hormones are blood, urine and saliva samples 2,3,4,5. Therefore, the aim of analytical laboratories is to develop a new, relatively low-cost and rapid implementation methodology for their determination in biological samples. Human serum samples were found to be stable for up to 14 days when stored in a refrigerator and 30 days in a freezer (Supplementary Figure S5). A standard from a different lot was purchased to prepare quality control samples at the concentrations of 35, 100, and 600 ng/dL. The results from blood drawn in serum-separator tubes (BD, Franklin Lakes, NJ, USA) and clot-activator tubes (red-top) were compared to determine the acceptability of serum separator tubes. The LOQ was the minimum concentration at which the CV was within 20%. For between-day studies, four samples were extracted in triplicate each day for ten days. Calibration standards were prepared in ACN at concentrations ranging from 0.05 to 2000 ng/mL, with 10–50 ng/mL of isotopically labelled internal standards, and were derivatized by following the procedure described above. Calibration curves of all analytes were constructed both in solvent and in fortified urine and serum matrices. The mobile phase flow was diverted to waste during the first 3.5 min after sample injection. Although chiral separation could eliminate the interference between 11ß-MNT and testosterone as demonstrated in A and B, broad chromatographs limit measurement sensitivity. The imperfection of this chiral separation method was that both analytes had relatively broad peaks in their chromatogram. While 11ß-MNT and testosterone could not be separated with a short retention time using reverse-phase chromatography, separation of the isomers could be achieved with short retention time when an appropriate chiral column was used . These mixtures were centrifuged for 15 minutes at 18,000 g to facilitate separation of the liquid phases, and the organic solvent phase was collected. These calibration standards and QC samples were prepared fresh each day, and standards were analyzed in duplicate; QC samples were analyzed in triplicate on each day of analysis. Zarzycki, P.K., Zarzycka, M.B. Application of temperature-controlled micro planar chromatography for separation and quantification of testosterone and its derivatives. Retention properties of steroids were investigated across a whole range of binary mixtures such as methanol/water, acetonitrile/water, methanol/dichloromethane and acetone/hexane (0–100% v/v). It can be successfully be used for the analysis of testosterone undecanoate from Andriol® gelatin capsule. Neutralize the sample solution with 10 ml of base and acid then volume to the mark with diluent.
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