Investigation of HPLC-MS using a monolithic column to separate a diverse suite of steroids

A rapid method for profiling steroids with a wide range of polarity has been developed using high-performance liquid chromatography-mass spectrometry with a monolithic LC column. Steroids are detected using tandem mass spectrometry (MS(n)) with a quadrupole ion trap and quantified using testosterone-d(3) as the internal standard. The method is compared to two similar methods using a traditional particulate column in terms of number of steroids eluted, peak area reproducibility, limits of detection, and overall analysis time. The monolithic method elutes the steroids in a 20-min analysis time, whereas the particulate methods elute the steroids in 30 and 45 min, respectively. The monolithic column also allows for improved reproducibility (relative standard deviations from 5-23%, as opposed to 14-42% for the shorter particulate method) and lower limits of detection (typically 2-5 times lower) when compared to the particulate column. Finally, the method is evaluated with unextracted, spiked alligator plasma, giving responses within 80-90% of those expected for standards for all steroids tested (except androstenedione).