Solvents are crucial for the separation analysis. Due to a variety of causes (such as toxicity, costly, unavailability, etc.), an analyst might seek to identify an alternative solvent since they can no longer utilize a solvent that they have relied on for many years. In search of the correct alternative solvent, the polarity model is a primary source to look at. The solvent selectivity triangle (SST), solvatochromic (ET(30) and ETN model), and Hildebrand polarity scale are the three polarity models which are primarily investigated to find acceptable alternative solvents with similar properties. All these models consist of three types of polarity components (i) acidity(α), (ii) basicity(β), and (iii) dipolarity /polarizbility(π*) and comprising polarity components that add up to the total polarity(P׳). In the comparison of the SST and solvatochromic model, further studies have identified the benefits of the solvatochromic parameter method of solvent polarity model over the solvent selectivity model. And thus, In this research, the solvatochromic ET(30) model has been applied for the analysis of the steroid isomer pairs.
Steroid isomers such as abiraterone metabolite pairs, estradiol isomers, bile acid enantiomer pairs have been playing a great role in the diagnostic of cancer treatment, female reproductive diseases, and metabolic regulations respectively. Estradiol is a steroid hormone produced largely by the ovaries during the reproductive lifespan and is essential for the development and the maintenance of the female reproductive system. Abiraterone is a steroidal compound with antiandrogen activity used to treat metastatic, castration-resistant prostate cancer and Bile acids have long been known to facilitate digestion and absorption of lipids in the small intestine as well as regulate cholesterol homeostasis. Research has shown that separation of the steroid isomers will create a scope to closely look and examine the isomers effectiveness in clinical treatment; for an example: new research shows that abiraterone is converted to a metabolite that has a wider range of inhibitory activity and clinically effective in the treatment of patients with prostate cancer than abiraterone itself but before the clinical analysis they need to be separated.
Because of having structural chemical similarities, carryover issues, coelution etc. the steroid isomers are tremendously difficult to separate.
The liquid chromatography separation and mass spectrometric detection were achieved by employing the Shimadzu UFLC system coupled to an LCMS 2020 single quadrupole mass spectrometer. Chromatographic separation was performed using an Agilent Zorbax Eclipse Plus C18, 150 mm x 2.1 mm, 3.5 µm (Agilent, Santa Clara, CA) column at a flow rate of 0.2 mL/min applying an ESI ionization source under the column temperature at 40 ℃. The isocratic mobile phase consisted of 0.1% formic acid in 100% water for mobile phase A and 0.1% formic acid in organic modifier concentrations for mobile phase B. The binary pump system was utilized experimentally to accurately mix the concentration of the components of the mobile phase delivered in an isocratic method, so the first peak could be obtained at the target retention time for a particular abiraterone pair for all mobile phase compositions. The selectivity factor was calculated for each run and the result was averaged. Triplicate injections using each type of the ten mobile phase compositions were carried out to ensure reproducibility. The column hold-up time was determined by injecting 10 μL of 0.5% formic acid in a 100% acetonitrile sample at a flow rate of 0.2 mL/min into a 100% acetonitrile mobile phase. The void time was found to be 1.92 minutes from the average of triplicate injections. Retention factors were measured at increments of 10% (v/v) of one of the organic solvents in the mixture for the composition range 0–100 % (v/v) methanol and isopropanol.
In this work, abiraterone metabolite pairs, and estradiol isomer pairs and bile acid isomers are well separated by applying the ternary mobile phase systems (consisting of water and two different organic modifiers) by utilizing isocratic reversed-phase LC-MS and LC-UV techniques using the C18 column instead of chiral column. The basis of the work was to keep the solvent strength constant such as 8 minutes or 15 minutes and thus it was possible to elute the first isomer of all isomer pairs at the target minutes using the ternary solvent system.
In this research, it has been found that the steroid isomer pairs showed optimized separation in reversed-phase liquid chromatography within short analysis time with greater selectivity. The graphs of selectivity factor versus ET(30) polarity component values were drawn to investigate the correlation of this model with the selectivity. It was found that the selectivity factor data of both steroid pairs were found to be best fitted with the ET(30) model. This research is beneficial in the clinical field for the analyst to choose a quick alternative solvent for the rapid separation of the steroid isomer pairs before moving for the close examination of the clinical analysis.