A sensitive and selective LC-differential mobility-mass spectrometric analysis of allopregnanolone and pregnanolone in human plasma

A sensitive and selective method was developed to quantitate allopregnanolone and its 5 beta isomer pregnanolone in human plasma using liquid chromatography-differential mobility separation combined with MS/MS detection. The method employed a simple liquid-liquid extraction of 100 mu L plasma with hexane/ethyl acetate. After extraction, the sample was derivatized using a quaternary aminooxy reagent. Separation of allopregnanolone, pregnanolone, and their 3 beta epimers (epiallopregnanolone and epipregnanolone) was achieved using a Phenomenex Kinetex C18 2.1 x 100-mm 2.6-mu m column. A linear calibration curve was obtained over the concentration range from 10 to 25,000 pg/mL, and the inter- and intra-day accuracy of the quality control samples were between 90 and 110 % with the inter- and intra-day precision less than 10 %. The lower limit of quantitation is 50 fg (157 amol) on column for both allopregnanolone and pregnanolone which is 100-fold less than the underivatized compounds. The recovery is above 95 %, and the extracted samples are stable for at least 6 days when stored at 4 A degrees C. Plasma samples from normal, pregnant, and postpartum women were analyzed using this method.