Advances in Steroid Panel Analysis with High Sensitivity LC/MS/MS. Kenneth C. Lewis 1, Lisa St. John-Williams 1, Changtong Hao 2, Sha Joshua Ye 2

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1 Advances in Steroid Panel Analysis with High Sensitivity LC/MS/MS Kenneth C. Lewis 1, Lisa St. John-Williams 1, Changtong Hao 2, Sha Joshua Ye 2 1 OpAns LLC, Durham, NC, USA; 2 Ionics Mass Spectrometry Group, Bolton, ON, Canada Introduction Steroid hormones regulate metabolism, neurotransmission, intracellular signaling and much more. Changes in the levels of these hormones produce a wide range of clinical effects in the areas of sedation, seizure prevention, oncology and reproduction. Quantification of multiple steroids from a single biological fluid sample is useful for both understanding diseases and for clinical treatment. Background We have previously developed assays for the simultaneous quantification of up to fourteen steroids from plasma. Achieving adequate sensitivity across the whole panel is quite difficult because optimum ionization conditions vary depending on the steroid. For example, the optimum source conditions for delta-5 steroids (e.g. pregnenolone) are much different than for cortisol. Achieving clinically relevant sensitivity for the whole panel with one set of conditions is exceptionally challenging. Steroid hormone concentrations are critical biomarkers for the understanding and diagnosis of disease. Steroid synthesis involves multiple routes and is not a sequential linear process. Understanding disease requires an understanding of the exact perturbation in the steroid synthesis pathway. We are most interested in the following abbreviated pathway.

2 The H295R cell line was developed as a model of steroidogenesis and is used as an in-vitro assay to determine if a chemical will disrupt the endocrine system. Measurement of terminal steroids (e.g. testosterone and estradiol) will indicate the system has been disturbed. However, other steroids need to be measured to determine exactly how the chemical interferes with steroidogenesis. The following plot shows the impact of the antifungal compound ketoconazole as a function of concentration in the H295R cell media.

3 Our desire is to provide a complete picture of steroidogenesis by quantifying all relevant steroid hormones in a single sample (in-vitro model system, in-vivo model system, or human clinical sample) Methods Steroids were extracted from 75 ml of media via a liquid-liquid process. The organic layer was removed, evaporated to dryness. Solutions of dansyl chloride and buffer were added to the dry container, sealed and heated. 7 ul of the sample was injected into a Fused Core C18 (Halo, Poroshell, Kinetex) 2.1 x 50 mm, 2.7mm column. 6 minutes gradient LC program was used with 0.5 ml/min flow rate using H 2 O with 0.1% formic acid and MeOH with 0.1% formic acid solvents. Shimadzu 10ADvp HPLC pumps and IONICS 3Q Series 200 Molecular Analyzer were used in this analysis. This method has the sensitivity to measure pg/ml levels of steroids from biological samples. This level of sensitivity can be achieved for androgens, glucocorticoids, and progestagens together in one analytical run; thereby enabling the analysis of a panel of steroids by one method. We have successfully used the 3Q to analyze H295R cell culture media for this panel of steroids over the physiological relevant ranges.

4 Analysis Conditions Instrumentation: LC: Shimadzu 10ADvp HPLC pumps Autosampler: CTC PAL MS: IONICS 3Q Series 200 Molecular Analyzer LC Conditions Solvent A: Solvent B: H2O with 0.1% formic acid MeOH with 0.1% formic acid Column: Fused Core C18 (Halo, Poroshell, Kinetex) 2.1 x 50 mm, 2.7 µm Injection Vol: 7 µl Flowrate: 0.5 ml/min MS/MS Conditions Gradient: Time (min) %B (stop) Ionization: ESI (positive ion) Source Parameters: ESI Voltage: 5500 V HSID Temp. 350 o C Probe Temp. 350 o C Drying Gas Setting 120 Neb Gas Setting 350

5 Heating Gas Setting 350 Detection: Multiple Reaction Monitoring Transitions: Cortisol m/z 363>121 Corticosterone m/z 347> deoxycortisol m/z 347>109 Androstenedione m/z 287>97 DOC m/z 331>109 Testosterone m/z 289>97 17OH-progesterone m/z 331>109 DHEA m/z 271> OH-pregnenolone m/z 315>297 progesterone m/z 315>109 pregnenolone m/z 299>281 dansyl-estrone m/z 504>171 dansyl-estradiol m/z 506>171 Results:

6 Area 1.E+08 1.E DeoxyCortisol R² = Androstenedione Area 1.E+07 R² = Area DOC R² = E Area 1.E+07 R² = Testosterone

7 Area Dansyl-Estradiol R² = E [Insert Captions and labels for figures] Conclusions The IONICS 3Q Series 200 Molecular Analyzer has the sensitivity to measure pg/ml levels of steroids from biological samples. This sensitivity can be achieved for androgens, glucocorticoids, and progestagens together in one analytical run; thereby enabling the analysis of a panel of steroids by one method. We have successfully analyzed the H295R cell culture media for this panel of steroids over the physiologically relevant ranges. Acknowledgements Special thanks to Colleen Toole and Hillary Wagner, our collaborators at CeeTox for performing all of the cell incubations.