We developed a method for the quantitative analysis of neuroactive steroids and their precursors using negative chemical ionization gas chromatography/mass spectrometry (GC/MS) that allows for the simultaneous detection of 8 steroids: 3alpha-hydroxy,5alpha-pregnan-20-one (3a,5a-THP), 3alpha-hydroxy,5beta-pregnan-20-one (3a,5b-THP), 3alpha,21-dihydroxy,5alpha-pregnan-20-one (3a,5a-THDOC), 3alpha, 21-dihydroxy,5beta-pregnan-20-one (3a,5b-THDOC), 3alpha-hydroxy,5alpha-androstan-17-one (3a,5a-A), 3alpha-hydroxy,5beta-androstan-17-one (3a,5b-A), and their precursors pregnenolone and dehydroepiandrosterone (DHEA). Deuterium labeled 3a,5a-THP, 3a,5a-THDOC, 3a,5a-A and pregnenolone were used as internal standards. Steroids were purified using two consecutive solid phase extraction procedures with C18 and NH2 cartridges and were derivatized with heptafluorobutyric acid anhydride. GC/MS was operated in selective ion monitoring mode, allowing for picograms (pg) of neuroactive steroid to be quantified. Standard curves were linear from 10 to 3000 pg. To validate the procedure, human stripped serum was spiked with known amounts of each steroid from 30 to 3000 pg and the steroid levels detected were compared to the amounts added to stripped serum. Linear regressions of the actual values compared to the expected values were R2 = 0.999 for 3a,5a- and 3a,5b-THP, 3a,5a- and 3a,5b-THDOC and 3a,5b-A and R2 = 0.998 for 3a,5a-A, pregnenolone and DHEA, showing the accuracy and validity of the assay. We tested the effect of progesterone administration in 8 healthy women during the follicular phase of the menstrual cycle. Blood was collected 15 minutes before and 160 minutes after oral administration of micronized progesterone (300 mg). Basal steroid levels (ng/ml ± SEM) were: 2.46 ± 0.57 for 3a,5a-THP, 8.89 ± 4.89 for 3a,5b-THP? 0.26 ± 0.11 for 3a,5a-THDOC, 0.50 ± 0.12 for 3a,5b-THDOC, 2.92 ± 1.02 for 3a,5a-A, 0.67 ± 0.22 for 3a,5b-A, 4.14 ± 0.93 for pregnenolone and 6.89 ± 0.88 for DHEA. Progesterone administration induced a significant increase in 3a,5a- and 3a,5b-THP serum levels (+1093%, p=0.0003 and +151%, p=0.041, respectively). Although 3a,5a-THDOC was not detected in 3/8, and 3a,5b-THDOC was not detected in 4/8 subjects, levels of both steroids were increased after progesterone administration. Levels of pregnenolone, 3a,5a-A and 3a,5b-A did not change, while DHEA levels were decreased by 36% (p=0.022). The present method allows simultaneous quantification of 8 neuroactive steroids in human serum and can be employed to study the role of neuroactive steroids in physiological and pathological conditions. Supported by AA 10564 and AA 016677.
Quantification of GABAergic neuroactive steroids and their precursors in human serum using negative chemical ionization gas chromatography/mass spectrometry
Porcu P;
2007
Abstract
We developed a method for the quantitative analysis of neuroactive steroids and their precursors using negative chemical ionization gas chromatography/mass spectrometry (GC/MS) that allows for the simultaneous detection of 8 steroids: 3alpha-hydroxy,5alpha-pregnan-20-one (3a,5a-THP), 3alpha-hydroxy,5beta-pregnan-20-one (3a,5b-THP), 3alpha,21-dihydroxy,5alpha-pregnan-20-one (3a,5a-THDOC), 3alpha, 21-dihydroxy,5beta-pregnan-20-one (3a,5b-THDOC), 3alpha-hydroxy,5alpha-androstan-17-one (3a,5a-A), 3alpha-hydroxy,5beta-androstan-17-one (3a,5b-A), and their precursors pregnenolone and dehydroepiandrosterone (DHEA). Deuterium labeled 3a,5a-THP, 3a,5a-THDOC, 3a,5a-A and pregnenolone were used as internal standards. Steroids were purified using two consecutive solid phase extraction procedures with C18 and NH2 cartridges and were derivatized with heptafluorobutyric acid anhydride. GC/MS was operated in selective ion monitoring mode, allowing for picograms (pg) of neuroactive steroid to be quantified. Standard curves were linear from 10 to 3000 pg. To validate the procedure, human stripped serum was spiked with known amounts of each steroid from 30 to 3000 pg and the steroid levels detected were compared to the amounts added to stripped serum. Linear regressions of the actual values compared to the expected values were R2 = 0.999 for 3a,5a- and 3a,5b-THP, 3a,5a- and 3a,5b-THDOC and 3a,5b-A and R2 = 0.998 for 3a,5a-A, pregnenolone and DHEA, showing the accuracy and validity of the assay. We tested the effect of progesterone administration in 8 healthy women during the follicular phase of the menstrual cycle. Blood was collected 15 minutes before and 160 minutes after oral administration of micronized progesterone (300 mg). Basal steroid levels (ng/ml ± SEM) were: 2.46 ± 0.57 for 3a,5a-THP, 8.89 ± 4.89 for 3a,5b-THP? 0.26 ± 0.11 for 3a,5a-THDOC, 0.50 ± 0.12 for 3a,5b-THDOC, 2.92 ± 1.02 for 3a,5a-A, 0.67 ± 0.22 for 3a,5b-A, 4.14 ± 0.93 for pregnenolone and 6.89 ± 0.88 for DHEA. Progesterone administration induced a significant increase in 3a,5a- and 3a,5b-THP serum levels (+1093%, p=0.0003 and +151%, p=0.041, respectively). Although 3a,5a-THDOC was not detected in 3/8, and 3a,5b-THDOC was not detected in 4/8 subjects, levels of both steroids were increased after progesterone administration. Levels of pregnenolone, 3a,5a-A and 3a,5b-A did not change, while DHEA levels were decreased by 36% (p=0.022). The present method allows simultaneous quantification of 8 neuroactive steroids in human serum and can be employed to study the role of neuroactive steroids in physiological and pathological conditions. Supported by AA 10564 and AA 016677.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
