Urinary steroid profiles in pregnancy by capillary column gas chromatography

A qualitative and quantitative method for the estimation of urinary steroids during pregnancy by capillary column gas chromatography has been developed. Steroid conjugates were hydrolysed by enzymes and free steroids isolated by extraction using SEP-PAK C18 cartridges. Methoxime-trimethylsilyl derivatives of the steroids were characterized by packed column gas chromatography-mass spectrometry and subsequently by their retention indices on capillary column gas chromatography. Of the 48 peaks resolved on the profiles about 35 were identified as steroids. The retention indices of reference steroids were very reproducible in the short term showing a mean coefficient of variation of 0.015%. The columns allowed 95% valley resolution of steroids whose retention indices differed by only 7 units. The use of an on-column injection technique contributed to the high precision for replicate injections (coefficient of variation less than 1%) while for reference steroids, and well-resolved peaks in the urine profile, the mean coefficient of variation for the complete assay was less than 10%. To the best of our knowledge this is the first report concerning the quantitation, with good precision, of a large number of steroids in pregnancy urine.