B-186 Investigating LC Column Selectivity for 19 Steroids in a Clinical Research Setting

Abstract

Abstract Background Steroids are a class of structurally related endogenous compounds. Several have the same chemical formula (and hence the same m/z) and must be chromatographically separated for accurate identification and quantitation by LC-MS/MS. Here, six bonded silica solid core or “core-shell” LC columns (see Table 1) were evaluated to determine selectivity and optimum separation conditions for a 19 analyte steroid panel for clinical research. The optimum column for the separation of estrone and estradiol was also established. Methods Analytical reference standards (Cerilliant®, Round Rock, TX) were used to prepare diluted samples for injection. Six different core-shell 2.6 µm, 50 × 2.1 mm LC columns (Kinetex™, Phenomenex, Torrance, CA) were evaluated (see Table 1). Detection employed a 7500 triple quadrupole LC-MS/MS equipped with an ESI source used in positive and negative mode (SCIEX®, Framingham, MA) coupled to an Agilent® 1290 liquid chromatograph (Agilent Technologies, Santa Clara, CA). Results The traditional C18 column worked best for the complete steroid clinical research panel. Estrogens had the best separation on the biphenyl phase, which demonstrated the highest level of selectivity for estrone and estradiol. The phenyl-hexyl column did not sufficiently separate all steroid isomers in the clinical research panel, however it was sufficient to separate estrone and estradiol. Conclusion The proposed HPLC conditions and the core-shell 2.6 μm 50 × 2.1 mm C18 column provided the best separation for 19 steroids with a fast 8 minute run time. For research focused on estrogens, the core-shell biphenyl column yielded the best separation.