Aim To establish a
method for simultaneously determination of dehydroisoandrosterone (DHEA),pregnenolone (PREG) and allopregnenolone(AP) in
rat plasma by HPLC/MS.Method Neurosteroids were isolated separately in a two-step procedure using ethyl acetate /heanxe(9∶1,v/v) in the first step to extract the unconjugated steroids,then steroid fractions were further purified by SPE.All steroids were derivatized with 2-nitro-4-trifluoromethylphenylhydrazine(2NFPH),After derivation,the concentration of DHEA,PREG and
AP were determined by liquid chromatograph/Negative atmosphere pressure chemical ionization-mass spectrometry.Methyltestosterone (MT) was used as the internal standard.Results The linear ranges of DHEA,PREG and AP were 0.030～2.00 ng·ml~(-1) (r=0.998 8),0.025～2.00ng·ml~(-1) (r=0.999 6,0.998 8) respectively.The extraction recoveries of plasma samples with low,middle and high concentration were ranged from 70.4% to 92.4%.The method recoveries were ranged from 92.6% to 105.6%.The RSD of intra-day and inter-day were less than 15%.The concentration of
preg and AP in normal SD rat plasma were 0.29±0.24ng·ml~(-1) and 0.19±0.20ng·ml~(-1) respectively.The concentration of DHEA was less than LOQ.Conclusion The method is sensitive,accurate,reproducible and could be used for determination of concentration of DHEA,PREG and AP in rat's plasma.
More abstracts about the Determination of Unconjugated Neurosteroids in Rat Plasma by Liquid Chromatography/negative Atomsphe