Steroid biosynthesis is a complex metabolic pathway utilizing simple precursors to synthesize multiple steroidal forms. This biosynthetic pathway is unique to animals and provides a common target for antibiotics and other anti-infective drugs. Precise and accurate steroid analysis is critical for the development of steroid-based therapeutics. Typical analysis methods utilize GC–MS, which requires sample derivatization and lengthy analysis times (~ 25 min), or LC–MS, with typical analysis times of 4–12 min. Many of the steroid structures are closely related making their analysis challenging even when using the selectivity of mass spectrometric detection. Chromatographic separation is therefore essential for analysis of steroids and steroid derivatives, resulting in long analysis times. Convergence chromatography, with CO2 as the primary mobile phase, presents a unique opportunity to provide rapid and precise analyses for these structurally related compounds (Figure 1).
Figure 1: Steroid structures for the current investigation.
A mixture of nine steroids was prepared at a concentration of 0.2 mg/mL each, using a diluent of 88:12 methanol/ethanol. Steroids used were: androstenedione, estrone, 17α-hydroxyprogesterone, testosterone, 11-deoxycortisol, estradiol, corticosterone, aldosterone, and cortisol.
All data was collected on an ACQUITY UltraPerformance Convergence Chromatography™ (UPC2™) system with PDA detection. The steroid sample was screened on three different ACQUITY UPC2™ column chemistries; BEH (P/N 186006562), BEH 2-EP (P/N 186006580), and CSH Fluoro-Phenyl (P/N 186006571), using a 1.7 μm particle size in a 3.0 × 50 mm column dimension. The mobile phases were CO2 with methanol as a co-solvent. A 2-min screening gradient was used from 2% to 17% methanol at a flow rate of 3.65 mL/min and a temperature of 40 °C. The automatic back-pressure regulator (ABPR) was set to 1800 psi. Data was collected at 220 nm (compensated for 380–480 nm). The injection volume was 1 μL.
The chromatograms shown in Figure 2 demonstrate the selectivity differences of the ACQUITY UPC2 stationary phases, as well as the inherent speed of this chromatographic technique, with a significant reduction in analysis times compared to alternative techniques. Without the need for derivitization (required for GC analysis), samples can be analyzed directly in organic extraction solvents, omitting the need for diluent exchange for compatibility with reversed-phase LC methods. These factors combined yield a streamlined work flow with significant savings in analysis and sample prep time, solvent cost, and solvent waste generated.
Figure 2: UPC2 separations (UV) of steroid standards on ACQUITY UPC2 columns: BEH (top), BEH 2-EP (middle), and CSH Fluoro-Phenyl (bottom). All columns were 1.7 Î¼m, 3.0 Ã 50 mm configurations. Steroid compounds are androstenedione (1), estrone (2), 17Î±-hydroxyprogesterone (3), testosterone (4), 11-deoxycortisol (5), estradiol (6), corticosterone (7), aldosterone (8), and cortisol (9).
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