Quantification of progesterone and 17-β estradiol in mouse serum by liquid chromatography-tandem mass spectrometry Authors: Benjamin Kennard, Allison Cobble, Amy Gravitte, Keleigh Galloway, Jen Kintner, Jennifer Hall, Stacy Brown Introduction: In the United States, Chlamydia trachomatis is a commonly appearing sexually transmitted infection1. It affects the U.S. healthcare system to a tune of about $500 million dollars annually2. In women, it generally appears asymptomatic and can lead to severe secondary complications such as pelvic inflammatory diseases or infertility1. Female sex hormones, estrogen and progesterone, are being identified to have a role in chlamydial infection. Specifically, this study aims to create quantification methods to detect levels of estrogen and progesterone in mice, infected with Chlamydia muridarum, plasma samples. Methods: Progesterone samples were prepared using solid-liquid extraction (SLE+) cartridges with ethyl acetate as the elution solvent. Estradiol samples were prepared using liquid-liquid extraction (LLE) with methyl tert-butyl ether and subsequent derivatization with DMIS. Following sample preparation, hormones were quantified in samples using LC-MS/MS with a gradient elution of 1 mM ammonium fluoride in water and acetonitrile. The separation was achieved using a UCT C18 column (100 x 21.mm, 1.8 μm particle size) maintained at 50oC. The mass spectrometer was set up to isolate molecular ions for progesterone (m/z 315.0910) and derivatized estradiol (m/z 431.1835). Quantification was facilitated by the use of deuterium-labeled internal standards and their corresponding molecular ions in the mass spectrometer (d9-progesterone; m/z 324.1230 and d5-estradiol; m/z 436.2922). Results: Several aspects of the assay presented have been optimized for maximum analyte recovery and analytical sensitivity, including column choice, mobile phase, derivatizing agents for estradiol, and extraction protocols for progesterone. The LC-MS/MS method was investigated for precision and accuracy over three separate days. The dynamic range of the progesterone assay was 5 – 100 ng/mL, with a limit of detection of 1 ng/mL. Likewise, the estradiol assay was linear in the range of 5 – 100 ng/mL, with a limit of detection of 0.5 ng/mL. The average precision, represented by % RSD was 0.74 – 8.5% and 6.3 – 13.4% for progesterone and estradiol, respectively. The accuracy of the method, represented by % error was 1.6 – 14.4% and 4.0 – 10.5% for progesterone and estradiol, respectively. Successful validation was defined as < 15% RSD and error (< 20% at the limit of quantification), per current FDA Guidelines. Conclusions: The developed LC-MS/MS method is specific for progesterone and estradiol, and the extraction is suitable for preparation of mouse serum samples. This assay could be successfully applied to hormone quantification in mouse samples to support the investigation of the link between chlamydia infection and hormone levels in female animals. References 1. Chlamydia - 2017 Sexually Transmitted Diseases Surveillance. https://www.cdc.gov/std/stats17/chlamydia.htm. Accessed October 23, 2018. 2. Owusu-Edusei K, Chesson HW, Gift TL, et al. The Estimated Direct Medical Cost of Selected Sexually Transmitted Infections in the United States, 2008. Sex Transm Dis. 2013;40(3):197-201. doi:10.1097/OLQ.0b013e318285c6d2