A quantitative method for the determination of allopregnanolone (5alpha,3alpha-THP) and related neurosteroids in CSF and plasma was established using gas chromatography/electron capture negative chemical ionization mass spectrometry (GC/ECNCI/MS). Neurosteroids were converted to carboxymethoxime, pentafluorobenzyl and trimethylsilyl derivatives and detected as intense (M-181)(-) fragment ions generated under the negative ion chemical ionization process. The response curves constructed using d(4)-dihydrotestosterone (DHT) and d(4)-5alpha,3alpha-THP as internal standards showed linearity in the concentration range of 10-1000 pg/ml. The variation of response ratios determined against internal standards over a 2-month period was less than 10%. Instrumental detection limits for most neurosteroids were in the low picogram range with the exception of progesterone and dihydroprogesterone (DHP) which were detected with approximately 10 times less sensitivity in comparison to other steroids. In conjunction with solid-phase extraction, this method allowed the quantification of at least four neurosteroids, including androsterone, testosterone, 5alpha,3alpha-THP, and pregnenolone in 1-2 ml of human cerebrospinal fluid (CSF). While the level of 5alpha, 3alpha-THP in human CSF was comparable to that in the human plasma, other steroid levels were significantly lower. Although individual CSF and plasma samples showed widely varying neurosteroid levels, species specificity appeared to exist. The levels of 5alpha, 3alpha-THP and pregnenolone in human CSF were higher than those of monkey CSF where these steroids were often not detected with our current detection limit. In comparison to human plasma, rat plasma samples contained considerably lower levels of androsterone and pregnenolone. Among THP stereoisomers, 5beta,3alpha-THP and 5alpha, 3beta-THP were observed only in human plasma, while 5beta,3beta-THP was detected only in rat plasma.
Copyright 2000 Academic Press.