Assessment of the benefits of adding differential mobility spectrometry to LC-MS/MS workflows for challenging steroid...

Accurate quantification of steroid hormones such as estrogens (estradiol, estrone) and glucocorticoids (cortisol, cortisone) is essential for diagnosing and monitoring endocrine disorders. However, their structural similarity and low physiological concentrations pose analytical challenges in clinical laboratories. This study evaluates the utility of differential mobility spectrometry (DMS) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for improving the specificity and sensitivity of steroid measurements in serum (estrogens) and scalp hair (glucocorticoids) using a SelexION-equipped Sciex 6500+ mass spectrometer coupled to a high throughput Thermo Scientific TLX-2 LC system. DMS significantly reduced chromatographic interferences and enhanced signal-to-noise (S/N) ratios-up to 420 % for estradiol and 210 % for estrone. For cortisol and cortisone in hair, DMS also reduced interferences and increased S/N, as well as improving fragment ion agreement as evidenced by the reduction in fragment ion calculated concentration discrepancies exceeding ±20 % from 18 to 8 samples for cortisol and from 23 to 2 samples for cortisone. These findings support DMS as a complementary technique to LC-MS/MS, offering orthogonal separation and improved analytical performance for steroid hormone quantification in complex biological matrices.