A Sensitive and Selective LC-Ion Mobility-Mass Spectrometric Analysis of Allopregnanolone and its Isomers in Human Plasma or Serum

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 130-162-Neuroendocrinology
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-157
Michael J . Y. Jarvis*1, Wen Jin1 and Margaret Altemus2
1AB Sciex, Concord, ON, Canada, 2Weill Cornell Medical College, New York, NY
For Research Use Only. Not For Use In Diagnostic Procedures. There is growing interest in the therapeutic potential of gabaergic neuroactive steroid compounds, and the 3-alpha metabolites of progesterone, testosterone, deoxycortisol and androstenedione have been shown to have potent anxiolytic, analgesic, antiseizure, and neuroprotective effects in animal models and to activate GABAA receptors. The most studied of these has been allopregnanolone. However, understanding of the physiological role of these compounds has been limited by the difficulty of measuring these compounds in biological samples. Currently only GC/MS assays with labor intensive extraction steps have adequate sensitivity to measure these compounds in biological samples and only a few specialized academic laboratories have the expertise to conduct these measurements. We propose to develop the capacity to use LC-MS/MS to measure GABAergic neurosteroid compounds in biological samples to enable the identification of biomarkers of disease risk, predictors of treatment response, and new therapeutic targets.

The challenges for LC-MS/MS analysis of allopregnanolone are (i) its poor ionization efficiency, and (ii) the presence of numerous isobaric interferences in biological samples including, but not limited to, its isomer pregnanolone. To overcome these challenges, ion mobility separation was combined with conventional LC-MS/MS detection using a highly sensitive AB SCIEX Triple Quad™ 6500 mass spectrometer equipped with the SelexION™ ion mobility device. The method employed liquid-liquid extraction of 100µL serum or plasma. After extraction, the sample was derivatized using a commercially available quaternary aminooxy reagent.

Separation of allopregnanolone and its isomer pregnanolone was achieved using a Phenomenex Kinetex C18 2.1x100 mm column. The calibration range was from 5 pg/mL to 100 ng/mL in serum or plasma, with inter- and intra-day precision less than 10% and inter- and intra-day accuracy between 90-110%. The recovery is 98%, and the limit of detection is 5 pg/mL for allopregnanolone and pregnanolone. Plasma samples from ‘normal’, pregnant, and postpartum women were analysed using this method.

Disclosure: MJYJ: Employee, AB SCIEX. WJ: Employee, AB SCIEX. Nothing to Disclose: MA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm